Tocopherols, tocotrienols, other chroman and side chain derivatives and uses thereof

ABSTRACT

The present invention provides an antiproliferative compound having a structural formula 
     
       
         
         
             
             
         
       
         
         
           
             where X and Y independently are oxygen, nitrogen or sulfur; R 1  is alkyl, alkenyl, alkynyl, aryl, heteroaryl, carboxylic acid, carboxylate, carboxamide, ester, thioamide, thiolacid, thiolester, saccharide, alkoxy-linked saccharide, amine, sulfonate, sulfate, phosphate, alcohol, ethers or nitriles; R 2  and R 3  are hydrogen or R 4 ; R 4  is methyl, benzyl carboxylic acid, benzyl carboxylate, benzyl carboxamide, benzylester, saccharide or amine; and R 5  is alkenyl; where when Y is nitrogen, said nitrogen is substituted with R 6 , wherein R 6  is hydrogen or methyl. Also provided are methods for treating a cell proliferative disease and for inducing apoptosis in a cell comprising administering this compound is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of applicationSer. No. 11/928,991, filed Oct. 30, 2007, now issued as U.S. Pat. No.7,718,814, which is a divisional of application Ser. No. 10/695,275,filed Oct. 28, 2003, now issued as U.S. Pat. No. 7,300,954 on Nov. 27,2007, which is a divisional of U.S. Ser. No. 10/008,066, filed Nov. 5,2001, now issued as U.S. Pat. No. 6,703,384 on Mar. 9, 2004, which is acontinuation-in-part of U.S. Ser. No. 09/502,592, filed Feb. 11, 2000,now issued as U.S. Pat. No. 6,770,672 on Aug. 3, 2004, which is acontinuation-in-part of U.S. Ser. No. 09/404,001, filed Sep. 23, 1999,now issued as U.S. Pat. No. 6,417,223 on Jul. 9, 2002, which claimsbenefit of priority to provisional application U.S. Ser. No. 60/101,542,filed Sep. 23, 1998, the entire contents of each of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of organicchemistry and antiproliferative and pro-apoptotic compounds. Morespecifically, the present invention relates to chroman-based compoundsand derivatives and analogs thereof, and their uses as cellanti-proliferative, proapoptotic, immunomodulating, and anti-canceragents.

2. Description of the Related Art

The regulatory controls of pro-life (cell proliferation) and pro-death(apoptosis) are extremely complex and involve multiple intracellularsignaling pathways and multiple interacting gene products. Cancer cellsmay exhibit multiple defects in normal regulatory controls of cellproliferation, such as enhanced expression of genes, which allow them toincrease in number. In addition to enhanced expression of pro-lifegenes, cancer cells down-regulate genes and their products that controlpro-death signals, resulting in the accumulation and potentialmetastasis of life threatening cancer cells. Thus, combinations ofunregulated cell proliferation and suppression of death inducingsignaling pathways give cancer cells both growth and survivaladvantages.

Whether a cell increases in numbers or not depends on a balance ofexpression of negatively-acting and positively-acting growth regulatorygene products, and the presence or absence of functional cell deathsignaling pathways. Negative-acting growth regulatory genes contributeto blockage of cells in the cell cycle. Positive-acting growthregulatory genes stimulate cells to progress through the cell cycle.Genes involved in apoptosis can be either proapoptotic or antiapoptotic,and the dynamic balance between them determines whether a cell lives ordies. Cancer cells, in order to survive and increase their numbers,undergo a series of mutational events over time that remove regulatorycontrols that give them the ability to grow unchecked and survive evenin the presence of proapoptotic signals, and develop attributes thatpermit them to escape detection and removal by the immune responsedefense system. Cancers may cause death of individuals unless removed bysurgery or effectively treated with drugs.

A wide variety of pathological cell proliferative conditions exist forwhich novel therapeutic strategies and agents are needed to providetherapeutic benefits. These pathological conditions may occur in almostall cell types capable of abnormal cell proliferation or abnormalresponsiveness to cell death signals. Among the cell types that exhibitpathological or abnormal growth and death characteristics are (1)fibroblasts, (2) vascular endothelial cells, and (3) epithelial cells.Thus, novel methods are needed to treat local or disseminatedpathological conditions in all or almost all organ and tissue systems ofindividuals.

Most cancers, whether they are male specific, such as prostate ortesticular, or female specific, such as breast, ovarian or cervical, or,whether they affect males and females equally, such as liver, skin orlung, with time undergo increased genetic lesions and epigenetic events,and eventually become highly metastatic and difficult to treat. Surgicalremoval of localized cancers has proven effective only when the cancerhas not spread beyond the primary lesion. Once the cancer has spread toother tissues and organs, the surgical procedures must be supplementedwith other more specific procedures to eradicate the diseased ormalignant cells. Most of the commonly utilized supplementary proceduresfor treating diseased or malignant cells such as chemotherapy orbioradiation are not localized to the tumor cells and, although theyhave a proportionally greater destructive effect on malignant cells,often affect normal cells to some extent.

Some natural vitamin E compounds, and some derivatives of vitamin E havebeen used as proapoptotic and DNA synthesis inhibiting agents.Structurally, vitamin E is composed of a chromanol head and an alkylside chain. There are eight major naturally occurring forms of vitaminE: alpha (α), beta (β), gamma (γ), and delta (δ) tocopherols and α, β,γ, and δ tocotrienols. Tocopherols differ from tocotrienols in that theyhave a saturated phytyl side chain rather than an unsaturated isoprenylside chain. The four forms of tocopherols and tocotrienols differ in thenumber of methyl groups on the chromanol head (a has three, β and γ havetwo and δ has one).

RRR-α-tocopheryl succinate is a derivative of RRR-α-tocopherol that hasbeen structurally modified via an ester linkage to contain a succinylmoiety instead of a hydroxyl moiety at the 6-position of the chromanhead. This ester linked succinate moiety of RRR-α-tocopherol has beenthe most potent form of vitamin E affecting the biological actions oftriggering apoptosis and inhibiting DNA synthesis. This form of vitaminE induces tumor cells to undergo apoptosis, while having no apoptoticinducing effects on normal cells. The succinated form of vitamin E iseffective as an anticancer agent as an intact agent; however, cellularand tissue esterases that can cleave the succinate moiety, therebyconverting the succinate form of RRR-α-tocopherol to the freeRRR-α-tocopherol, render this compound ineffective as an anticanceragent. RRR-α-tocopherol exhibits neither antiproliferative norproapoptotic biological activity in cells of epithelial or immuneorigin.

The prior art is deficient in the lack of effective means of inhibitingundesirable or uncontrollable cell proliferation in a wide variety ofpathophysiological conditions while having no to little effect on normalcells. The present invention fulfills this long-standing need and desirein the art.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, there is provided a compoundhaving a structural formula

where X and Y independently are oxygen, nitrogen or sulfur; R¹ is alkyl,alkenyl, alkynyl, aryl, heteroaryl, carboxylic acid, carboxylate,carboxamide, ester, thioamide, thiolacid, thiolester, saccharide,alkoxy-linked saccharide, amine, sulfonate, sulfate, phosphate, alcohol,ethers or nitriles; R² and R³ are hydrogen or R⁴; R⁴ is methyl, benzylcarboxylic acid, benzyl carboxylate, benzyl carboxamide, benzylester,saccharide or amine; and R⁵ is alkenyl; where when Y is nitrogen, saidnitrogen is substituted with R⁶, wherein R⁶ is hydrogen or methyl.

In another embodiment of the present invention there is provided amethod for the treatment of a cell proliferative disease comprisingadministering to an individual a pharmacologically effective dose of acompound having a structural formula

where R² and R³ are hydrogen or R⁴; R⁴ is methyl, benzyl carboxylicacid, benzyl carboxylate, benzyl carboxamide, benzylester, saccharide oramine; and R⁵ is alkenyl; R⁷ is hydroxyl or —XR¹; wherein R¹ is alkyl,alkenyl, alkynyl, aryl, heteroaryl, carboxylic acid, carboxylate,carboxamide, ester, thioamide, thiolacid, thiolester, saccharide,alkoxy-linked saccharide, amine, sulfonate, sulfate, phosphate, alcohol,ethers or nitriles; and X and Y independently are oxygen, nitrogen orsulfur with the proviso that when Y is nitrogen, said nitrogen issubstituted with R⁶, wherein R⁶ is hydrogen or methyl.

In yet another embodiment of the present invention there is provided amethod of inducing apoptosis of a cell using the compounds disclosedsupra.

Other and further aspects, features, benefits, and advantages of thepresent invention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention are briefly summarized. above may be had by referenceto certain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted; however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1 shows general structure of tocopherol, tocotrienol and otherchroman-based compounds.

FIGS. 2A, 2B and 2C shows general synthetic organic approaches for thechemical variation of chromanol compounds at position R¹.

FIG. 3 shows general synthetic organic approaches for the chemicalvariation of chromanol compounds at position R².

FIG. 4 shows general synthetic organic approaches for the chemicalvariation of chromanol compounds at position R³ and R⁴.

FIG. 5 shows general synthetic organic approaches for the chemicalvariation of chromanol compounds at position R⁵.

FIGS. 6A-6B show general synthetic organic approaches for theall-racemic 1-aza-α-tocopherol analogs. FIG. 6A shows the syntheticscheme for compounds 31-38 and FIG. 6B shows the synthetic scheme forcompounds 39-43.

FIG. 7 shows the mean body weights of non-tumor Balb/c female mice atday 11 and day 23 of a maximum tolerated dose study.

FIGS. 8A-8C show the comparison of percent mean tumor weight followingtreatments in MDA MB-435 Human Breast Cancer Cells (FIG. 8A), DU-145Human Prostrate Cancer Cells (FIG. 8B) and HT-29 Human Colon CancerCells (FIG. 8C).

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention, there is provided a compoundhaving a structural formula

where X and Y independently are oxygen, nitrogen or sulfur; R¹ is alkyl,alkenyl, alkynyl, aryl, heteroaryl, carboxylic acid, carboxylate,carboxamide, ester, thioamide, thiolacid, thiolester, saccharide,alkoxy-linked saccharide, amine, sulfonate, sulfate, phosphate, alcohol,ethers or nitriles; R² and R³ are hydrogen or R⁴; R⁴ is methyl, benzylcarboxylic acid, benzyl carboxylate, benzyl carboxamide, benzylester,saccharide or amine; and R⁵ is alkenyl; where when Y is nitrogen, saidnitrogen is substituted with R⁶, wherein R⁶ is hydrogen or methyl.

In an aspect of this embodiment the compound has a formula where X and Yare oxygen and R⁵ is a C₇₋₁₆ olefinic group containing 3 to 5 ethylenicbonds. A representative example of this compound is 6-O-carboxymethylalpha-tocotrienol. Pharmaceutical compounds comprising the instantcompounds and an acceptable pharmaceutical carrier are also provided.

In another embodiment of the present invention there is provided amethod for the treatment of a cell proliferative disease comprisingadministering to an individual a pharmacologically effective dose of acompound having a structural formula

where R² and R³ are hydrogen or R⁴; R⁴ is methyl, benzyl carboxylicacid, benzyl carboxylate, benzyl carboxamide, benzylester, saccharide oramine; and R⁵ is alkenyl; R⁷ is hydroxyl or —XR¹; wherein R¹ is alkyl,alkenyl, alkynyl, aryl, heteroaryl, carboxylic acid, carboxylate,carboxamide, ester, thioamide, thiolacid, thiolester, saccharide,alkoxy-linked saccharide, amine, sulfonate, sulfate, phosphate, alcohol,ethers or nitriles; and X and Y independently are oxygen, nitrogen orsulfur with the proviso that when Y is nitrogen, said nitrogen issubstituted with R⁶, wherein R⁶ is hydrogen or methyl.

In an aspect of this embodiment, the compound has a formula where Y isoxygen, R⁷ is hydroxyl or —OR¹, and R⁵ is a C₇₋₁₆ olefinic groupcontaining 3 to 5 ethylenic bonds. Representative examples of thesecompounds are is α-tocotrienol, γ-tocotrienol, δ-tocotrienol, or6-O-carboxymethyl α-tocotrienol.

The method of this embodiment may be used to treat an animal. Mostpreferably, the methods of the present invention are useful in humans.Treatment may be effected by administering a pharmacologically effectivedose of the instant compounds of about 1 mg/kg to about 60 mg/kg.Administration of such a dose may be by oral, topical,liposomal/aerosol, intraocular, intranasal, parenteral, intravenous,intramuscular, or subcutaneous delivery. The instant compounds exhibitan anti-proliferative effect; representative examples of theseanti-roliferative effects are apoptosis, DNA synthesis arrest, cellcycle arrest, or cellular differentiation.

The compounds and methods of the present invention may be used to treatneoplastic diseases and non-neoplastic diseases. Representative examplesof neoplastic diseases are ovarian cancer, cervical cancer, endometrialcancer, bladder cancer, lung cancer, cervical cancer, breast cancer,prostate cancer, testicular cancer, gliomas, fibrosarcomas,retinoblastomas, melanomas, soft tissue sarcomas, osteosarcomas, coloncancer, carcinoma of the kidney, pancreatic cancer, basal cellcarcinoma, and squamous cell carcinoma. Representative examples ofnon-neoplastic diseases are selected from the group consisting ofpsoriasis, benign proliferative skin diseases, ichthyosis, papilloma,restenosis, scleroderma hemangioma, and leukoplakia.

The compounds and methods of the present invention may be used to treatnon-neoplastic diseases that develop due to failure of selected cells toundergo normal programmed cell death or apoptosis. Representativeexamples of diseases and disorders that occur due to the failure ofcells to die are autoimmune diseases. Autoimmune diseases arecharacterized by immune cell destruction of self cells, tissues andorgans. A representative group of autoimmune diseases includesautoimmune thyroiditis, multiple sclerosis, myasthenia gravis, systemiclupus erythematosus, dermatitis herpetiformis, celiac disease, andrheumatoid arthritis. This invention is not limited to autoimmunity, butincludes all disorders having an immune component, such as theinflammatory process involved in cardiovascular plaque formation, orultra violet radiation induced skin damage.

The compounds and methods of the present invention may be used to treatdisorders and diseases that develop due to viral infections.Representative examples of diseases and disorders that occur due toviral infections are human immunodeficiency viruses (HIV). Since thesecompounds are working on intracellular signaling networks, they have thecapacity to impact signal transduction of any type of external cellularsignal such as cytokines, viruses, bacteria, toxins, heavy metals, etc.

In yet another embodiment of the present invention there is provided amethod of inducing apoptosis of a cell using the compounds disclosedsupra. In an aspect of this embodiment the method may be used to treat acell proliferative disease.

The following definitions are given for the purpose of facilitatingunderstanding of the inventions disclosed herein. Any terms notspecifically defined should be interpreted according to the commonmeaning of the term in the art.

As used herein, the term “individual” shall refer to animals and humans.

As used herein, the term “biologically inhibiting” or “inhibition” ofthe growth of proliferating cells shall include partial or total growthinhibition and also is meant to include decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory doseof the composition of the present invention may be determined byassessing the effects of the test element on target malignant orabnormally proliferating cell growth in tissue culture, tumor growth inanimals and cell culture or any other method known to those of ordinaryskill in the art.

As used herein, the term “induction of programmed cell death orapoptosis” shall include partial or total cell death with cellsexhibiting established morphological and biochemical apoptoticcharacteristics. The dose of the composition of the present inventionthat induces apoptosis may be determined by assessing the effects of thetest element on target malignant or abnormally proliferating cell growthin tissue culture, tumor growth in animals and cell culture or any othermethod known to those of ordinary skill in the art.

As used herein, the term “induction of cell cycle arrest” shall includegrowth arrest due to treated cells being blocked in GO/G1 or G2/M cellcycle phase. The dose of the composition of the present invention thatinduces cell cycle arrest may be determined by assessing the effects ofthe test element on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell culture orany other method known to those of ordinary skill in the art.

As used herein, the term “induction of cellular differentiation” shallinclude growth arrest due to treated cells being induced to undergocellular differentiation as defined by established morphological andbiochemical differentiation characterization, a stage in which cellularproliferation does not occur. The dose of the composition of the presentinvention that induces cellular differentiation may be determined byassessing the effects of the test element on target malignant orabnormally proliferating cell growth in tissue culture, tumor growth inanimals and cell culture or any other method known to those of ordinaryskill in the art.

As used herein, the term “growth inhibitory concentration (IC₅₀)” or“effective concentration (EC₅₀)” shall include the effective therapeuticdose of a compound or composition for controlling cancer growth, i.e.,by blocking 50% cancer growth via DNA synthesis inhibition, cellulardifferentiation, cell cycle blockage and/or cell death.

Naturally occurring alpha-, gamma-, and delta tocotrienols, as well asgamma- and delta-tocopherols exhibit anticancer activity.Alpha-tocopherol does not exhibit anticancer activity. The presentinvention provides tocopherols, tocotrienols, and other chromanderivatives with or without derivatives of saturated phytyl orunsaturated isoprenyl side chains and analogs; e.g., azo- andthiol-analogs, thereof. Construction of RRR-alpha-tocopherol orRRR-alpha-tocotrienol based compounds with a succinate or modifiedsuccinate moiety attached to the C6 position of the first ring of thechromonal head of alpha-tocopherol or alpha-tocotrienol via an etherlinkage provides novel compounds with potent anticancer properties, forin vivo use, are produced. The lack of cellular etherases in mammalsinsures the efficacy of these compounds. The general structures of thenatural tocopherols and tocotrienols of the present invention are shownin FIG. 1, and possible routes for their syntheses are provided in FIGS.2-5.

These molecules include chemical funtionalization of positions R¹-R⁵ ofthe chroman structure, and chemical functionalization of the phytyl andisoprenyl side chains, particularly compounds based on tocopherols andtocotrienols. Additionally, compounds with heteroatom substitutions (Nor S) for the chroman ring oxygen are presented (FIG. 6A-6B).

The pharmacodynamically designed compounds of the present invention havean improved therapeutic index and are potent inhibitors of cancer cellgrowth; i.e., they demonstrate high antitumor activity with minimal sideeffects. These compounds, which can not be readily degraded since thereare no known etherases in mammals, may be used in the treatment ofcancers and disorders involving excess cell proliferation, as well asfor cells that accumulate in numbers due to suppressed cell killingmechanisms, with minimal side effects. The compounds of the presentinvention inhibit cancer cell growth by induction of celldifferentiation, induction of apoptosis and DNA synthesis arrest.Induction of apoptosis and, by extension, inhibition of tumor growth, bythese compounds is via modulation of the transforming growth factor-beta(TGF-β), Fas/Fas ligand, and certain mitogen-activated protein kinases(MAPK) signaling pathways, or, in the case of some tocotrienols, isexpected to involve these pathways. Induction of apoptosis via otherpathways, such as ceramide production, is not excluded. These growthinhibitory properties allow these compounds to be used in the treatmentof proliferative diseases, including cancers of different cell types andlineages, non-neoplastic hyperproliferative diseases, and disorders withdefects in apoptotic signaling pathways. Several of the compounds of thepresent invention are both strong inducers of apoptosis and stronginhibitors of DNA synthesis arrest of tumor cells representing differentcellular lineages.

The therapeutic use of the compounds of the present invention intreatment of cancers and other diseases and disorders involving excesscell proliferation or failure of cells to die is illustrated. The novelderivatives (Tables 2-1/2-2 and 3-1/3-2) are shown at EC₅₀concentrations to induce apoptosis of human breast cancer cells (MDA MB435, MDA MB 231, and MCF-7 breast cancer cells), human prostate cancercells (PC-3, DU-145 and LnCaP), human ovarian tumor cells (C-170), humancervical tumor cells (ME-180), human endometrial cells (RL-95-2), humanlymphoid cells (myeloma, Raji, Ramos, Jurkat, and HL-60), colon cancercells (HT-29 and DLD-1) and lung cancer cells (A-549). The novelderivatives were shown to not induce apoptosis of normal human mammaryepithelial cells (HMECs) and immortalized but non-tumorigenic MCF-10Amammary cells.

Generally, to achieve pharmacologically efficacious cell killing andanti-proliferative effects, these compounds and analogs thereof may beadministered in any therapeutically effective dose. Preferably, thestructurally modified tocopherols and tocotrienols and analogs areadministered in a dose of from about 0.1 mg/kg to about 100 mg/kg. Morepreferably, the structurally modified tocopherols and tocotrienols andanalogs are administered in a dose of from about 1 mg/kg to about 10mg/kg.

Administration of the compounds and compositions of the presentinvention may be by liposome/aerosol, topical, intraocular, parenteral,oral, intranasal, intravenous, intramuscular, subcutaneous, or any othersuitable means. The dosage administered is dependent upon the age,clinical stage and extent of the disease or genetic predisposition ofthe individual, location, weight, kind of concurrent treatment, if any,and nature of the pathological or malignant condition. The effectivedelivery system useful in the method of the present invention may beemployed in such forms as liposomal aerosol, capsules, tablets, liquidsolutions, suspensions, or elixirs, for oral administration, or sterileliquid forms such as solutions, suspensions or emulsions. For topicaluse it may be employed in such forms as ointments, creams or sprays. Anyinert carrier is preferably used in combination with suitablesolubilizing agents, such as saline, or phosphate-buffered saline, orany such carrier in which the compounds used in the method, such asethanol, acetone, or DMSO, of the present invention have suitablesolubility properties.

There are a wide variety of pathological cancerous and noncancerous cellproliferative conditions and cell accumulations due to absence of normalcellular death for which the compositions and methods of the presentinvention will provide therapeutic benefits. These pathologicalconditions may occur in almost all cell types capable of abnormal cellproliferation or defective in programmed cell death mechanisms. Amongthe cell types which exhibit pathological or abnormal growth or abnormaldeath are (1) fibroblasts, (2) vascular endothelial cells and (3)epithelial cells. It can be seen from the above that the methods of thepresent invention is useful in treating local or disseminatedpathological conditions in all or almost all organ and tissue systems ofindividuals.

It is specifically contemplated that pharmaceutical compositions may beprepared using the novel chroman-based compounds and derivatives andanalogs thereof of the present invention. In such a case, thepharmaceutical composition comprises the novel compounds of the presentinvention and a pharmaceutically acceptable carrier. A person havingordinary skill in this art would readily be able to determine, withoutundue experimentation, the appropriate dosages and routes ofadministration of the compounds and analogs of the present invention.

Thus the present invention is directed toward the design and effectiveuse of novel agents that can specifically target cancer cells and eitherdown-regulate growth stimulatory signals, up-regulate growth inhibitorysignals, down-regulate survival signals and/or up-regulate deathsignals. More specifically, this invention creates and characterizesnovel agents that activate growth inhibitory factors, trigger deathsignaling pathways, and inhibit DNA synthesis.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE 1 Synthetic Organic Methodology

The synthesis of a variety of tocopherol, tocotrienol, and other chromanderivatives with or without derivatives of saturated phytyl orunsaturated isoprenyl side chains or analogs thereof is possible viastructural modification of the chroman ring system (FIGS. 2-5) andheteroatom substitutions (N or S) for the chroman ring oxygen (FIG.6A-6B). The structural variables R¹, R², R³, R⁴, R⁵, R⁷ and X illustratethe groups on the chroman group that are modified and Y representseither oxygen, or heteroatom substitutions (N or S) for the chroman ringoxygen. In naturally occurring α-, γ-, δ-tocopherols and α-, β-, γ-,δ-tocotrienols, the X—R¹ substituent at the six position of the chromanring is hydroxyl; in a general structure this is represented by R⁷. WhenY is nitrogen, it may be substituted with a structural variable R⁶,e.g., hydrogen or methyl.

Using alkylation chemistry, a large number of compounds containingdifferent R¹ groups can be synthesized, particularly when X is oxygen.After alkylation, further chemical modification of the R¹ groups permitsthe synthesis of a wide range of novel compounds. R1 substituents can bealkyl, alkenyl, alkynyl, aryl, heteroaryl, carboxylic acid, carboxylate,carboxamide, ester, thioamide, thiolacid, thiolester, saccharide,alkoxy-linked saccharide, amine, sulfonate, sulfate, phosphate, alcohol,ethers and nitriles.

Bromination of the benzylic methyl groups of the chroman group provideintermediates that permit variation of the R², R³ and R⁴ groups.Substituents for R² and R³ may be hydrogen or those substituents for R⁴,e.g., methyl, benzyl carboxylic acid, benzyl carboxylate, benzylcarboxamide, benzylester, saccharide and amine. Variation of group R⁵,such as alkyl, alkenyl, alkynyl, aryl, heteroaryl, carboxyl, amide andester, is also possible, particularly when starting from thecommercially available 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid.

When a heteroatom substitution of nitrogen for the chroman ring oxygenoccurs, the nitrogen may be substituted with R⁶ which is hydrogen ormethyl. Variation of X to groups other than oxygen, which is theidentity of X in tocopherols and tocotrienols, can be accomplished usingpalladium chemistry (for X═CH₂) and nucleophilic aromatic substitution(for X═N or S). Other possible modifications to the chroman structureinclude unsaturation at the 3-4 positions and ring contraction toproduce a five-membered furanyl ring.

Reagents employed were either commercially available or preparedaccording to a known procedure. Anhydrous CH₂Cl₂ and THF were obtainedby distillation. All other solvents used were reagent. Anhydrousreaction conditions were maintained under a slightly positive argonatmosphere in oven-dried glassware. Silica gel chromatography wasperformed using 230-400 mesh silica for tocopherol compounds and 70-200mesh silica for tocotrienol compounds purchased from EM Science. Routine¹H- and ¹³C-NMR spectra were obtained on a Varian Unity spectrometer at300.132 MHz and 75.033 MHz frequencies, respectively. NMR spectra werereferenced to TMS (0 ppm) or to the isotopic impurity peak of CDCl₃(7.26 and 77.0 ppm for ¹H and ¹³C, respectively). High resolutionelectron impact ionization mass spectroscopy was performed by the MassSpectrometry Center at The University of Texas at Austin.

EXAMPLE 2 Synthesis and characterization of tocopherol compounds2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)aceticacid (1)

A solution of R,R,R-α-tocopherol (0.5 g, 1.16 mmol) inN,N-dimethylformamide (20 mL) was treated with methyl bromo acetate (3.4g, 8.3 mmol) and an excess of powdered NaOH (1.2 g, 30 mmol). Theresulting yellow slurry was stirred vigorously for 24 h at roomtemperature. The reaction was acidified with 5 N HCl and extracted withdiethyl ether (3×30 ml). The combined ether layers were washed with H₂O(3×30 ml) and brine (1×30 ml), and then dried with Na₂SO₄. The ethersolution was concentrated to a yellow oil that was purified by silicagel chromatography eluting with 19% (v/v) EtOAc and 2% acetic acid inhexanes. The resulting yellow liquid was dissolved in diethyl ether (30ml), washed with H₂O (3×20 mL) and brine (1×20 mL), and then dried withNa₂SO₄. The resulting solution was concentrated to a light yellow oiland dried in vacuo for 48 h. This yielded 1 as a waxy, off-white solid(0.50 g, 88%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH, 1′-,2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂), 2.07, 2.14, 2.16(3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.59 (t, J=6.6 Hz, 2H, 4-CH₂), 4.34 (s, 2H,OCH₂); ¹³C-NMR (CDCl₃, ppm): 11.7, 11.8, 12.7 (5a-, 7a-, 8a-CH₃), 19.6,19.7 (CH₃), 20.6, 21.0 (CH₂), 22.6, 22.7 (CH₃), 23.8 (2a-CH₃), 24.4,24.8 (CH₂), 28.0 (CH), 31.2 (3-CH₂), 32.7, 32.8 (CH), 37.3, 37.4, 37.5,39.4, 40.0 (CH₂), 69.2 (OCH₂), 75.0 (2-C), 117.8, 123.2, 125.4, 127.3(aryl C), 147.0, 148.5 (aryl C—O), 173.7 (COOH); HRMS (Cl, m/z):489.394374 (M+H⁺, Calc. for C₃₁H₅₃O₄ 489.394386). All assignments wereconfirmed using HMQC, DEPT-135, and ¹H-NOSEY.

2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)propionic acid (2)

The compounds 2-6 are synthesized in a manner identical to the synthesisof 1 using the appropriate bromoalkanoic acids.

(89% yield). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂), 2.09, 2.14, 2.19(3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.59 (t, J=6.6 Hz, 2H, 4-CH₂), 2.85 (t,J=6.4 Hz, 2H, CH₂COOH), 3.96 (t, J=6.4 Hz, 2H, OCH₂); ¹³C-NMR (CDCl₃,ppm): 11.7, 11.8, 12.7 (5a-, 7a-, 8a-CH₃), 19.6, 19.7 (CH₃), 20.6, 21.0(CH₂), 22.6, 22.7 (CH₃), 23.8 (2a-CH₃), 24.4, 24.8 (CH₂), 28.0 (CH),31.2 (3-CH₂), 32.7, 32.8 (CH), 35.1, 37.3, 37.4, 37.5, 39.4, 40.0 (CH₂),67.5 (OCH₂), 74.8 (2-C), 117.5, 122.9, 125.8, 127.8 (aryl C), 147.6,148.0 (aryl C—O), 177.1 (COOH); HRMS (CI, m/z): 503.408610 (M+H⁺, Calc.for C₃₂H₅₅O₄ 503.410036).

2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)butyric acid (3)

(85% yield). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 26H, 4′-, 8′-, 12′-CH,1′-,2′-,3′-,5′-,6′-,7′-,9′-,10′-,11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂),2.14, 2.17, 2.21 (3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.62 (t, J=6.6 Hz, 2H,4-CH₂), 2.72 (t, J=7.2 Hz, 2H, CH₂COOH), 3.74 (t, J=6.1 Hz, 2H, OCH₂);¹³C-NMR (CDCl₃, ppm): 11.7, 11.8, 12.7 (5a-, 7a-, 8a-CH₃), 19.6, 19.7(CH₃), 20.6, 21.0 (CH₂), 22.6, 22.7 (CH₃), 23.9 (2a-CH₃), 24.4, 24.8,25.3 (CH₂), 28.0 (CH), 30.9, 31.2 (3-CH₂), 32.7, 32.8 (CH), 37.3, 37.4,37.5, 39.4, 40.0 (CH₂), 71.3 (OCH₂), 74.8 (2-C), 117.5, 122.9, 125.7,127.7 (aryl C), 147.8, 147.9 (aryl C—O), 178.9 (COOH); HRMS (CI, m/z):516.424374 (M+H⁺, Calc. for C₃₃H₅₇O₄ 516.424386).

2,5,7,8-tetramethyl-2R-(4R,8,R,12-trimethyltridecyl)chroman-6-yloxy)valeric acid (4)

(90% yield). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 28H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂), 2.09, 2.14, 2.18(3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.49 (t, J=6.8 Hz, 2H, CH₂COOH), 2.59 (t,J=6.6 Hz, 2H, 4-CH₂), 3.68 (t, J=5.5 Hz, 2H, OCH₂); ¹³C-NMR (CDCl₃,ppm): 11.7, 11.8, 12.7 (5a-, 7a-, 8a-CH₃), 19.6, 19.7 (CH₃), 20.6, 21.0,21.4 (CH₂), 22.6, 22.7 (CH₃), 23.8 (2a-CH₃), 24.4, 24.8 (CH₂), 28.0(CH), 30.0 (CH₂), 31.2 (3-CH₂), 32.7, 32.8 (CH), 35.8, 37.3, 37.4, 37.5,39.4, 40.0 (CH₂), 72.2 (OCH₂), 74.9 (2-C), 117.8, 123.2, 125.4, 127.3(aryl C), 147.6, 148.3 (aryl C—O), 178.7 (COOH); HRMS (CI, m/z):530.433514 (M+H⁺, Calc. for O₃₄H₅₉O₄ 530.433516).

2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)hexanoicacid (5)

(77% yield). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 30H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂), 2.08, 2.12, 2.16(3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.32 (t, J=6.5 Hz, 2H, CH₂COOH), 2.57 (t,J=6.6 Hz, 2H, 4-CH₂), 3.64 (t, J=5.5 Hz, 21-1, OCH₂); ¹³C-NMR (CDCl₃,ppm): 11.8, 11.9, 12.7 (5a-, 7a-, 8a-CH₃), 19.6, 19.7 (CH₃), 20.6, 21.0(CH₂), 22.6, 22.7 (CH₃), 23.8 (2a-CH₃), 24.4, 24.6, 24.8, 25.7 (CH₂),28.0 (CH), 30.0 (CH₂), 31.3 (3-CH₂), 32.7, 32.8 (CH), 34.0, 37.3, 37.3,37.4, 39.3, 40.0 (CH₂), 72.6 (OCH₂), 74.7 (2-C), 117.4, 122.7, 125.4,127.8 (aryl C), 147.6, 148.2 (aryl C—O), 179.6 (COOH); HRMS (CI, m/z):545.457026 (M+H⁺, Calc. for C₃₅H₆₁O₄ 545.456986).

2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)octanoic acid (6)

(91% yield). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 34H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH), 2.08, 2.11, 2.16(3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.36 (m, 2H, CH₂COOH), 2.58 (t, J=6.6 Hz,2H, 4-CH₂), 3.62 (t, J=5.5 Hz, 211, OCH₂); ¹³C-NMR (CDCl₃, ppm): 11.7,11.8, 12.7 (5a-, 7a-, 8a-CH₃), 19.6, 19.7 (CH₃), 20.6, 21.0 (CH₂), 22.6,22.7 (CH₃), 23.8 (2a-CH₃), 24.4, 24.6, 24.8, 25.1, 25.7, 26.6 (CH₂),28.0 (CH), 30.0 (CH₂), 31.3 (3-CH₂), 32.7, 32.8 (CH), 34.0, 37.3, 37.3,37.4, 39.3, 40.0 (CH₂), 72.7 (OCH₂), 74.6 (2-C), 117.6, 122.8, 125.5,127.6 (aryl C), 147.5, 148.3 (aryl C—O), 179.4 (COOH); HRMS (CI, m/z):573.484396 (M+H⁺, Calc. for C₃₇H₆₅O₄ 573.488286).

2,5,8-trimethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy) aceticacid (7)

A solution of R,R,R-α-tocopherol (75 mg, 0.18 mmol) inN,N-dimethylformamide (2 mL) was treated with methyl bromoacetate (0.4g, 2.8 mmol) and an excess of powdered NaOH (0.5 g, 12.5 mmol). Theresulting yellow slurry was stirred vigorously for 24 h at roomtemperature. The reaction was acidified with 5 N HCl and extracted withdiethyl ether (3×10 ml). The combined ether layers were washed with H₂O(3×10 ml) and brine (1×10 ml), and then dried with Na₂SO₄. The ethersolution was concentrated to a yellow oil that was purified by silicagel chromatography eluting with 19% (v/v) EtOAc and 2% acetic acid inhexanes. The resulting yellow liquid was dissolved in diethyl ether (30ml), washed with H₂O (3×10 mL) and brine (1×10 mL), and then dried withNa₂SO₄. The resulting solution was concentrated to a light yellow oiland dried in vacuo for 48 h. This yielded 7 as a waxy, off-white solid(80 mg, 97%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂), 2.12, 2.14 (2×s,6H, 5a-, 8a-CH₃), 2.61 (t, J=6.6 Hz, 2H, 4-CH₂), 4.59 (s, 2H, OCH₂),6.53 (s, 1H, aryl CH); ¹³C-NMR (CDCl₃, ppm): 11.2, 16.1 (5a-, 8a-CH₃),19.6, 19.7 (CH₃), 20.7, 21.0 (CH₂), 22.6, 22.7 (CH₃), 23.8 (2a-CH₃),24.4, 24.8 (CH₂), 27.9 (CH), 31.2 (3-CH₂), 32.7, 32.8 (CH), 37.2, 37.4,37.5, 39.4, 40.0 (CH₂), 66.8 (OCH₂), 74.8 (2-C), 113.8, 120.7, 123.1,127.3 (aryl C), 147.1, 148.2 (aryl C—O), 175.3 (COOH); HRMS (CI, m/z):475.377840 (M+H⁺, Calc. for C₃₀H₅₁O₄ 475.378736).

2,7,8-trimethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)aceticacid (8)

A solution of R,R,R-α-tocopherol (100 mg, 0.24 mmol) inN,N-dimethylformamide (5 mL) was treated with methyl bromoacetate (1.1g, 7.4 mmol) and an excess of powdered NaOH (1.0 g, 25 mmol). Theresulting yellow slurry was stirred vigorously for 24 h at roomtemperature. The reaction was acidified with 5 N HCl and extracted withdiethyl ether (3×10 ml). The combined ether layers were washed with H₂O(3×10 ml) and brine (1×10 ml), and then dried with Na₂SO₄. The ethersolution was concentrated to a yellow oil that was purified by silicagel chromatography eluting with 19% (v/v) EtOAc and 2% acetic acid inhexanes. The resulting yellow liquid was dissolved in diethyl ether (30ml), washed with H₂O (3×10 mL) and brine (1×10 mL), and then dried withNa₂SO₄. The resulting solution was concentrated to a light yellow oiland dried in vacuo for 48 h. This yielded 8 as a waxy, off-white solid(110 mg, 97%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂), 2.12, 2.19 (2×s,6H, 7a-, 8a-CH₃), 2.61 (t, J=6.6 Hz, 2H, 4-CH₂), 4.59 (s, 2H, OCH₂),6.39 (s, 1H, aryl CH); ¹³C-NMR (CDCl₃, ppm): 11.9, 12.0 (7a-, 8a-CH₃),19.6, 19.7 (CH₃), 20.7, 21.0 (CH₂), 22.6, 22.7 (CH₃), 23.8 (2a-CH₃),24.4, 24.8 (CH₂), 27.9 (CH), 31.2 (3-CH₂), 32.7, 32.8 (CH), 37.2, 37.4,37.5, 39.4, 40.0 (CH₂), 66.6 (OCH₂), 75.7 (2-C), 110.6, 117.7, 125.0,126.3 (aryl C), 146.9, 148.7 (aryl C—O), 175.0 (COOH); HRMS (CI, m/z):475.377962 (M+H⁺, Calc. for C₃₀H₅₁O₄ 475.378736).

2,8-dimethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)acetic acid(9)

A solution of R,R,R-α-tocopherol (100 mg, 0.25 mmol)N,N-dimethylformamide (5 mL) was treated with methyl bromoacetate (1.1g, 7.4 mmol) and an excess of powdered NaOH (1.0 g, 25 mmol). Theresulting yellow slurry was stirred vigorously for 24 h at roomtemperature. The reaction was acidified with 5 N HCl and extracted withdiethyl ether (3×10 ml). The combined ether layers were washed with H₂O(3×10 ml) and brine (1×10 ml), and then dried with Na₂SO₄. The ethersolution was concentrated to a yellow oil that was purified by silicagel chromatography eluting with 19% (v/v) EtOAc and 2% acetic acid inhexanes. The resulting yellow liquid was dissolved in diethyl ether (30ml), washed with H₂O (3×10 mL) and brine (1×10 mL), and then dried withNa₂SO₄. The resulting solution was concentrated to a light yellow oiland dried in vacuo for 48 h. This yielded 9 as a waxy, off-white solid(111 mg, 98%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH, 2′-, 3′-, 5′-, 6′-, 7′-,9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₁), 2.15 (s, 3H, 8a-CH₃),2.71 (t, J=6.6 Hz, 2H, 4-CH₂), 4.59 (s, 2H, OCH₂), 6.48 (d, J=3.0 Hz,1H, aryl CH), 6.61 (d, J=3.0 Hz, 1H, aryl CH); ¹³C-NMR (CDCl₃, ppm):16.2 (8a-CH₃), 19.6, 19.7 (CH₃), 21.0 (CH₂), 22.6, 22.7 (CH₃), 24.0(2a-CH₃), 24.4, 24.8 (CH₂), 27.9 (CH), 31.2 (3-CH₂), 32.7, 32.8 (CH),37.2, 37.4, 37.5, 39.4, 40.0 (CH₂), 65.7 (OCH₂), 75.8 (2-C), 112.3,115.6, 121.1, 127.5 (aryl C), 147.2, 149.9 (aryl C—O), 174.8 (COOH);HRMS (CI, m/z): 460.3552022 (M+H⁺, Calc. for C₃₀H₅₁O₄ 460.355262).

2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)acetamide (10)

A solution of 1 (0.1 g, 0.2 mmol) in CH₂Cl₂ (5 mL) was treated withN-hydroxysuccinimide (26 mg, 0.23 mmol) and dicyclohexylcarbodiimide (46mg, 0.23 mmol). After 2 min, a white precipitate formed. The resultingsuspension was stirred for 2 h. The reaction stirred for an additional 6h. The reaction mixture was cooled to −30° C. and filtered. The filtratewas concentrated and the resulting colorless oil was purified by silicagel chromatography eluting with EtOAc (35%, v/v) in hexanes. Thisyielded a white solid (75 mg, 76%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m,12H, 4a′-, 8a′-, 12a′-, 13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH,1′-, 2′-, 3′-, 5′-, 6′-, 7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H,3-CH₂), 2.10, 2.12, 2.16 (3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.59 (t, J=6.6 Hz,2H, 4-CH₂), 4.19 (s, 2H, OCH₂), 6.36, 6.92 (2× broad, 211, NH); ¹³C-NMR(CDCl₃, ppm): 11.7, 11.8, 12.7 (5a-, 7a-, 8a-CH₃), 19.6, 19.7 (CH₃),20.6, 21.0 (CH₂), 22.6, 22.7 (CH₃), 23.8 (2a CH₃), 24.4, 24.8 (CH₂),28.0 (CH), 31.2 (3-CH₂), 32.7, 32.8 (CH), 37.3, 37.4, 37.5, 39.4, 40.0(CH₂), 70.9 (OCH₂), 74.9 (2-C), 117.8, 123.3, 125.4, 127.3 (aryl C),146.5, 148.4 (aryl C—O), 172.1 (COOH); HRMS (CI, m/z): 488.409341 (M+H⁺,Calc. for C₃₁H₅₄NO₃ 488.410370).

Methyl2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)acetate(11)

A solution of 1 (0.1 g, 0.2 mmol) in CH₂Cl₂ (5 mL) was treated withN,N-dimethylaminopyridine (26 mg, 0.23 mmol), methanol (1 ml) anddicyclohexylcarbodiimide (46 mg, 0.23 mmol) After 2 min, a whiteprecipitate formed. The resulting suspension was stirred for 6 h. Thereaction mixture was cooled to −30° C. and filtered. The filtrate wasconcentrated and the resulting colorless oil was purified by silica gelchromatography eluting with EtOAc (40%, v/v) in hexanes. This yielded awhite solid (82 mg, 80%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-,8a′-, 12a′-, 13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-,5′-, 6′-, 7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂), 2.10,2.16, 2.20 (3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.59 (t, J=6.6 Hz, 2H, 4-CH₂),3.85 (s, 3H, OCH₃), 4.32 (s, 2H, OCH₂); ¹³C-NMR (CDCl₃, ppm): 11.7,11.8, 12.7 (5a-, 7a-, 8a-CH₃), 19.6, 19.7 (CH₃), 20.6, 21.0 (CH₂), 22.6,22.7 (CH₃), 23.8 (2a-CH₃), 24.4, 24.8 (CH₂), 28.0 (CH), 31.2 (3-CH₂),32.7, 32.8 (CH), 37.3, 37.4, 37.5, 39.4, 40.0 (CH₂), 50.2 (OCH₃), 69.8(OCH₂), 74.9 (2-C), 117.6, 123.0, 125.6, 127.5 (aryl C), 147.6, 148.2(aryl C—O), 169.8 (COOH); HRMS (CI, m/z): 503.408-411 (M+H⁺, Calc. forC₃₂H₅₅O₄ 503.410036).

2-(N,N-(carboxymethyl)-2(2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)acetic acid (12)

A solution of 1 (0.2 g, 0.4 mmol) in CH₂Cl₂ (5 mL) was treated withdiethyl iminodiacetate (77 mg, 0.4 mmol) andO-7-azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) (46 mg, 0.23 mmol). After 12 h, the reaction mixturewas concentrated to a paste and then purified by silica gelchromatography eluting with EtOAc (30%, v/v) in hexanes. This yieldedthe desired diester intermediate as colorless oil (150 mg, 55%). ¹H-NMR(CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-, 13′-CH₃), 1.0-1.6 (m,30H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-, 7′-, 9′-, 10′-, 11′-CH₂,2a-CH₃), 1.78 (m, 2H, 3-CH₂), 2.08, 2.13, 2.17 (3×s, 9H, 5a-, 7a-,8a-CH₃), 2.58 (t, J=6.8 Hz, 2H, 4-CH₂), 4.19, 4.22 (q, J=7.4 Hz, 4H,OCH₂), 4.30, 4.33, 4.42 (3×s, 6H, 2×NCH₂, OCH₂); ¹³C-NMR (CDCl₃, ppm):11.7, 11.8, 12.7 (5a-, 7a-, 8a-CH₃), 14.0 (CH₃), 19.6, 19.7 (CH₃), 20.6,21.0 (CH₂), 22.6, 22.7 (CH₃), 23.8 (2a-CH₃), 24.4, 24.8 (CH₂), 28.0(CH), 31.2 (3-CH₂), 32.7, 32.8 (CH), 37.3, 37.4, 37.5, 39.4, 40.0 (CH₂),48.1, 49.4 (NCH₂), 61.2, 61.5 OCH₂), 71.8 (OCH₂), 74.8 (2-C), 117.5,122.9, 125.6, 127.4 (aryl C), 148.0, 148.1 (aryl C—O), 168.8, 169.0(CO); MS (CI, m/z): 660 (M+H⁺, Calc. for C₃₉H₆₅NO₇ 659.47610).

A solution of the diester intermediate (0.15 g, 0.23 mmol) in ethanol (4ml) was treated with 1 N NaOH (1 ml). The resulting cloudy mixture wasstirred at 70° C. for 15 h. The reaction mixture was acidified with 1 NHCl and the ethanol was removed in vacuo. The resulting aqueous solutionwas extracted with CHCl₃ (5×20 ml) and the combined organic layers driedwith Na₂SO₄. This yielded 12 (0.13 g, 52%) as a white solid. ¹H-NMR(CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-, 13′-CH₃), 1.0-1.6 (m,24H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-, 7′-, 9′-, 10′-, 11′-CH₂,2a-CH₃), 1.70 (m, 2H, 3-CH₂), 2.01, 2.05, 2.08 (3×s, 9H, 5a-, 7a-,8a-CH₃), 2.47 (m, 2H, 4-CH₂), 4.18 (m, 4H, 2×NCH₂), 4.31 (m, 2H, OCH₂);¹³C-NMR (CDCl₃, ppm): 11.5, 11.6, 12.4 (5a-, 7a-, 8a-CH₃), 19.4, 19.5(CH₃), 20.6, 21.0 (CH₂), 22.6, 22.7 (CH₃), 23.8 (2a-CH₃), 24.4, 24.8(CH₂), 28.0 (CH), 31.2 (3-CH₂), 32.4, 32.5 (CH), 37.0, 37.2, 37.5, 39.1,40.0 (CH₂), 48.1, 49.4 (NCH₂), 71.1 (OCH₂), 74.8 (2-C), 117.5, 122.9,125.4, 127.2 (aryl C), 147.8, 148.1 (aryl C—O), 168.8, 169.0 (CO); HRMS(CI, m/z): 604.420882 (M+H⁺, Calc. for C₃₅H₅₈NO₇ 604.421329).

2-(2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy))ethan-1-ol(13)

A solution of R,R,R-α-tocopherol (0.5 g, 1.16 mmol) inN,N-dimethylformamide (20 mL) was treated with iodoethanol (1.7 g, 10mmol) and an excess of powdered NaOH (2.5 g, 63 mmol). The resultingyellow slurry was stirred vigorously for 24 h at room temperature. Thereaction was acidified with 5 N HCl and extracted with diethyl ether(3×30 ml). The combined ether layers were washed with H₂O (3×30 ml) andbrine (1×30 ml), and then dried with Na₂SO₄. The ether solution wasconcentrated to a yellow oil that was purified by silica gelchromatography eluting with 30% (v/v) EtOAc and 2% acetic acid inhexanes. The resulting yellow liquid was dissolved in diethyl ether (30ml), washed with H₂O (3×20 mL) and brine (1×20 mL), and then dried withNa₂SO₄. The resulting solution was concentrated to a light yellow oiland dried in vacuo for 48 h. This yielded 13 as yellow oil (0.40 g,73%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂), 2.07, 2.14, 2.16(3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.59 (t, J=6.6 Hz, 2H, 4-CH₂), 3.79 (m, 2H,OCH₂), 3.94 (m, 2H, OCH₂); ¹³C-NMR (CDCl₃, ppm): 11.7, 11.8, 12.7 (5a-,7a-, 8a-CH₃), 19.6, 19.7 (CH₃), 20.6, 21.0 (CH₂), 22.6, 22.7 (CH₃), 23.8(2a-CH₃), 24.4, 24.8 (CH₂), 28.0 (CH), 31.2 (3-CH₂), 32.7, 32.8 (CH),37.3, 37.4, 37.5, 39.4, 40.0 (CH₂), 63.1, 69.2 (OCH₂), 75.0 (2-C),117.8, 123.4, 126.4, 128.3 (aryl C), 149.2, 149.5 (aryl C—O); MS (CI,m/z): 475 (M+H⁺, Calc. for C₃₁H₅₄O₃ 474.40729).

2-(2,5,7,8-pentamethylchroman-6-yloxy)acetic acid (14)

A solution of 2,2,5,7,8-pentamethyl-6-chromanol (0.3 g, 1.36 mmol) inN,N-dimethylformamide (20 mL) was treated with methyl bromoacetate (0.8g, 5.3 mmol) and an excess of powdered NaOH (0.7 g, 18 mmol). Theresulting yellow slurry was stirred vigorously for 24 h at roomtemperature. The reaction was acidified with 5 N HCl and extracted withdiethyl ether (3×30 ml). The combined ether layers were washed with H₂O(3×30 ml) and brine (1×30 ml), and then dried with Na₂SO₄. The ethersolution was concentrated to a yellow oil that was purified by silicagel chromatography eluting with 30% (v/v) EtOAc and 2% acetic acid inhexanes. The resulting yellow liquid was dissolved in diethyl ether (30ml), washed with H₂O (3×20 mL) and brine (1×20 mL), and then dried withNa₂SO₄. The resulting solution was concentrated to a light yellow oiland dried in vacuo for 48 h. This yielded 14 as a white solid (0.31 g,82%). ¹H-NMR (CDCl₃/TMS, ppm): 1.31 (s, 6H, CH₃), 1.81 (t, J=7.8 Hz,3-CH₂), 2.10, 2.16, 2.19 (3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.61 (t, J=7.8 Hz,2H, 4-CH₂), 4.39 (s, 2H, OCH₂); ¹³C-NMR (CDCl₃, ppm): 11.7, 11.8, 12.7(5a-, 7a-, 8a-CH₃), 20.9, 26.8, 32.7 (alkyl), 69.1, (OCH₂), 72.9 (2-C),117.5, 123.2, 125.5, 127.3 (aryl), 147.6, 148.6 (O-aryl), 173.8 (COOH);HRMS (CI, m/z): 279.159238 (M+H⁺, Calc. for C₁₆H₂₃O₄ 279.159634).

2,5,7,8-tetramethyl-(2RS-(4RS,8RS,12-trimethyltridcyl)chroman-6-yloxy)aceticacid (15)

A solution of all racemic-α-tocopherol (0.5 g, 1.16 mmol) inN,N-dimethylformamide (20 mL) was treated with methyl bromoacetate (3.4g, 8.3 mmol) and an excess of powdered NaOH (1.2 g, 30 mmol). Theresulting yellow slurry was stirred vigorously for 24 h at roomtemperature. The reaction was acidified with 5 N HCl and extracted withdiethyl ether (3×30 ml). The combined ether layers were washed with H₂O(3×30 ml) and brine (1×30 ml), and then dried with Na₂SO₄. The ethersolution was concentrated to a yellow oil that was purified by silicagel chromatography eluting with 19% (v/v) EtOAc and 2% acetic acid inhexanes. The resulting yellow liquid was dissolved in diethyl ether (30ml), washed with H₂O (3×20 mL) and brine (1×20 mL), and then dried withNa₂SO₄. The resulting solution was concentrated to a light yellow oiland dried in vacuo for 48 h. This yielded 15 as a waxy, off-white solid(80%). ¹H-NMR (CDCl₃/TMS, ppm): 0.88 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.84 (m, 2H, 3-CH₂), 2.07, 2.14, 2.16(3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.61 (t, J=6.6 Hz, 2H, 4-CH₂), 4.34 (s, 2H,OCH₂); ¹³C-NMR (CDCl₃, ppm): 11.5, 11.7, 12.6 (5a-, 7a-, 8a-CH₃), 19.6,19.7 (CH₃), 20.6, 21.3 (CH₂), 22.6, 22.8 (CH₃), 23.8 (2a-CH₃), 24.5,24.9 (CH₂), 29.0 (CH), 31.6 (3-CH₂), 32.6, 32.8 (CH), 37.5, 37.8, 37.9,39.5, 41.0 (CH₂), 69.3 (OCH₂), 75.1 (2-C), 117.9, 123.3, 125.5, 127.3(aryl C), 147.0, 148.0 (aryl C—O), 173.9 (COON); HRMS (CI, m/z):489.394375 (M+H⁺, Calc. for C₃₁H₅₃O₄ 489.394383).

2,5,7,8-tetramethyl-(2R-(carboxy)chroman-6-yloxy))acetic acid (16)

A solution of (−)-(R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid (0.34 g, 1.36 mmol) in N,N-dimethylformamide (20 mL) was treatedwith methyl bromoacetate (0.8 g, 5.3 mmol) and an excess of powderedNaOH (0.7 g, 18 mmol). The resulting yellow slurry was stirredvigorously for 24 h at room temperature. The reaction was acidified with5 N HCl and extracted with diethyl ether (3×30 ml). The combined etherlayers were washed with H₂O (3×30 ml) and brine (1×30 ml), and thendried with Na₂SO₄. The ether solution was concentrated to a yellow oilthat was purified by silica gel chromatography eluting with 30% (v/v)EtOAc and 2% acetic acid in hexanes. The resulting yellow liquid wasdissolved in diethyl ether (30 ml), washed with H₂O (3×20 mL) and brine(1×20 mL), and then dried with Na₂SO₄. The resulting solution wasconcentrated to light yellow oil and dried in vacuo for 48 h. Thisyielded 16 as a white solid (0.33 g, 80%). ¹H-NMR (CDCl₃/TMS, ppm): 1.52(s, 3H, 2a-CH₃), 2.10 (m, 2H, 3-CH₂), 2.12, 2.16, 2.19 (3×s, 9H, 5a-,7a-, 8a-CH₃), 2.56 (t, J=6.5 Hz, 2H, 4-CH₂), 4.36 (s, 2H, OCH₂).

2,5,7,8-tetramethyl-2R-(2RS,6RS,10-trimethylundecyl)chroman-6-yloxy)aceticacid (17)

A solution of 10 g (40 mmol) of(−)-(S)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid and 0.5 gof p-toluenesulfonic acid monohydrate in 200 ml of methanol was stirredand refluxed for 4 hr. After cooling, the solution was diluted withwater and extracted with diethyl ether. The combined ether layers werewashed with saturated aqueous sodium bicarbonate solution, H₂O, andbrine (1×30 ml), and then dried with Na₂SO₄. The resulting solution wasconcentrated and dried in vacuo for 48 h. This yielded 10 g (95%) ofmethyl (−)-(S)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate as acolorless solid which was used without further purification. ¹H-NMR(CDCl₃/TMS, ppm): 1.52 (s, 3H, 2a-CH₃), 2.10 (m, 2H, 3-CH₂), 2.12, 2.16,2.19 (3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.56 (t, J=6.5 Hz, 2H, 4-CH₂), 3.55(s, 3H, OCH₃); MS (CI, m/z): 264.422 M+H⁺, Calc. for C₁₅H₂₀O₄ 265.3224.

To a solution of 2 g (7.58 mmol) of this ester in 7.5 ml of N,N-dimethylformamide (DMF) was added 2.6 g (18.8 mmol) of anhydrousgranular potassium carbonate followed by 2.3 ml (20 mmol) ofbenzylchloride. The resulting slurry was stirred at RT for 41 h thenpoured into 50 ml of water and worked up with ether in the usual way.The product was freed of excess benzyl chloride at 50° under highvacuum. There was obtained 2.69 g (100%) of pure (TLC)(−)-(S)-6-benzyloxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid methylester as a yellow solid, m.p. 102-106°. The analytical specimen of thiscompound was prepared as a colorless solid m.p. 108-109° (fromether/methanol). ¹H-NMR (CDCl₃/TMS, ppm): 1.54 (s, 3H, 2a-CH₃), 2.01 (m,2H, 3-CH₂), 2.14, 2.17, 2.19 (3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.51 (t, J=6.7Hz, 2H, 4-CH₂), 3.64 (s, 3H, OCH₃), 5.12 (s, 2H, 6-OCH₂), 7.15 (m, 5H,ArH); MS (CI, m/z): 355.232 M+H⁺, Calc. for C₂₂H₂₅O₄ 354.448.

A solution of 3.54 g (10 mmol) of the above ether ester, in 20 ml oftoluene and 10 ml of CH₂Cl₂ was stirred with cooling from dryice/acetone bath while 12 ml (18 mmol) of 25% disobutylaluminum hydridein toluene (Texas Alkyls) was added dropwise, over 10 min. Afterstirring at ca. −70° for 30 min, the reaction mixture was cautiouslydecomposed (−70°) with 10 ml of MeOH. Following the addition of 50 ml ofwater and 50 ml of 1N aqueous H₂SO₄ solution, the mixture was warmed toRT, and worked up with ether in the usual way giving 3.2 g (100%) ofcrude aldehyde [(+)S-6-Benzyloxy-2,5,7,8-tetramethylchroman-2-carbaldhyde] as a viscous oilwhich was purified by silica gel chromatography eluting with 19% (v/v)EtOAc in hexane. ¹H-NMR (CDCl₃/TMS, ppm): 1.53 (s, 3H, 2a-CH₃), 2.11 (m,2H, 3-CH₂), 2.24, 2.27, 2.29 (3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.481 (t,J=6.7 Hz, 2H, 4-CH₂), 5.19 (s, 2H, 6-OCH₂), 7.20 (m, 5H, ArH), 9.6 (s,1H, CHO); MS (CI, m/z): 325.332 M+H⁺, Calc. for C₂₁H₂₄O₃ 324.422.

A solution of 9.6 g of pseudoionone was dissolved in 100 ml of 95%ethanol; after 0.68 g of sodium borohydride in ethanol had been added atroom temperature, the mixture was stirred for 2 hr and then leftstanding overnight. The mixture was added to a solution of 2 g of sodiumhydroxide in 500 ml of water. The mixture was extracted with ether, andthe ether extract was washed with water, dried, and concentrated. Thedistillation of the residual oil in vacuo gave a colorless oil(pseudoionol); by 112-120° C./5 mmHG. 7.7 g (80%).

To a solution of 2.97 g of pseudoionol in 10 ml of acetonitrile, therewere added, under stirring and while the temperature was kept below 30°C., 4.53 g of triphenylphospine hydrochloride which had been obtained bypassing dry hydrogen chloride into a solution of triphenylphosphine indry ether. After the mixture had been left standing overnight at roomtemperature, the acetonitrile was removed under reduced pressure below50° C. To the residue there were added 4.47 gm of (+)S-6-Benzyloxy-2,5,7,8-tetramethylchroman-2-carbaldhyde in 15 ml ofdimethylformamide, and the mixture was stirred. When a clear solutionwas obtained, sodium methoxide prepared from 0.352 g of sodium and 7 mlof anhydrous methanol was stirred in, drop by drop below 15° C. Thereaction mixture was turned red by the ylid formed. After the additionwas complete, stirring was continued for 30 min at 10° C.; then themixture was gradually heated to 80° C., when the red color disappeared.The product was poured into 200 ml of 50% aqueous methanol, dried, andconcentrated in vacuo. The residual oil was dissolved in 20 ml of ether,and an etheral solution of mercuric chloride was added until no moreprecipitate formed. When the precipitate was filtered and the filtratewas washed with water, dried and concentrated, to give 4.7 g of yellowoil were obtained. The crude mixture of cis and trans alkene (MS (CI,m/z): 485.22, M+H⁺, Calc. for C₃₄H₄₄O₂ 484.7255) was dissolved in 30 mlof ethyl acetate and 0.80 g of 5% palladium on carbon was added, and themixture was shaken under 40 psi of H, for 30 hrs and then filteredthrough Celilte and rinsed well with ethyl acetate. The filtrate wasconcentrated and purified by silica gel chromatography eluting withEtOAc in hexane (1:9) to give2,5,7,8-tetramethyl-(2R-(2RS,6RS,10-trimethylundecyl))-6-chromanol (60%yield) ¹H-NMR (CDCl₃/TMS, ppm): 0.97 (m, 12H, 2a′-, 6a′-, 10a′-,11′-CH₃), 1.1-1.7 (m, 20H, 2′-, 6′-, 10′-CH, 3′-4′-, 5′-, 7′-, 8′-,9′-CH₂, 2a-CH₃), 1.88 (m, 2H, 3-CH₂), 2.17, 2.19, 2.20 (3×s, 9H, 5a-,7a-, 8a-CH₃), 2.63 (t, J=6.7 Hz, 2H, 4-CH₂); (MS (CI, m/z): 403.27,M+H⁺, Calc. for C₂₇H₄₆O₂ 402.6632.

A solution of2,5,7,8-tetramethyl-(2R-(2RS,6RS,10-trimethylundecyl))-6-chromanol(0.466 g, 1.16 mmol) in N,N-dimethylformamide (20 mL) was treated withmethyl bromoacetate (3.4 g, 8.3 mmol) and an excess of powdered NaOH(1.2 g, 30 mmol). The resulting yellow slurry was stirred vigorously for24 h at room temperature. The reaction was acidified with 5 N HCl andextracted with diethyl ether (3×30 ml). The combined ether layers werewashed with H₂O (3×30 ml) and brine (1×30 ml), and then dried withNa₂SO₄. The ether solution was concentrated to a yellow oil that waspurified by silica gel chromatography eluting with 19% (v/v) EtOAc and2% acetic acid in hexanes. This yielded compound 17 in 76% yield. ¹H-NMR(CDCl₃/TMS, ppm): 0.97 (m, 12H, 2a′-, 6a′-, 10a′-, 11′-CH₃), 1.2-1.7 (m,20H, 2′-, 6′-,10′-CH, 3′-, 4′-, 5′-, 7′-, 8′-, 9′-CH₂, 2a-CH₃), 1.92 (m,2H, 3-CH₂), 2.18, 2.20, 2.23 (3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.68 (t, J=6.8Hz, 2H, 4-CH₂), 4.48 (s, 2H, OCH₂); MS (CI, m/z): 461.44, M+H⁺, Calc.for C₂₉H₄₈O₄ 460.700.

2,5,7,8-tetramethyl-2R-(2,6,10-trimethyl-1,3,5,9 E:Z decatetraen)chroman-6-yloxy)acetic acid (18)

To a solution of methyl(−)-(S)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (20 gms 0.075mole) in 50 ml of dry DMF, imidazole (13 gm, 0.1911 mole), andtert-butyldimethyl-silyl chloride (14 gm, 0.0933 mole) were added. Themixture was stirred at 23° C. for 24 hr and then treated with ether andpoured into 1N HCl. The organic extracts were dried (brine, Na₂SO₄) andconcentrated in vacuo. The crude product was purified by flashchromatography (9:1 hexane:ethyl acetate) to yield6-[dimethyl(1,1-dimethylethyl)silyl]-2,5,7,8-tetramethyl-chroman-2-carboxylate(TBS protected methyl ester). ¹H-NMR (CDCl₃/TMS, ppm): 0.12 (s, 6H).1.102 (s, 9H), 1.18 (s, 3H), 1.48 (s, 3H), 1.645 (s, 3H), 2.07 (s, 3H),2.2 (t, J=6.5 hz 2H), 2.48-2.7 (m, 2H) and 3.72 (s, 3H, OCH₃) (MS (CI,m/z): 379.32, M+H⁺, Calc. for C₂₁H₃₄O₄ 378.586.

A solution of 3.78 g (10 mmol) of the above ether ester, in 20 ml oftoluene and 10 ml of CH₂Cl₂ was stirred with cooling from dryice/acetone bath while 12 ml (18 mmol) of 25% disobutylaluminum hydridein toluene (Texas Alkyls) was added dropwise, over 10 min. Afterstirring at ca. −70° for 30 min, the reaction mixture was cautiouslydecomposed (−70°) with 10 ml of MeOH. Following the addition of 50 ml ofwater and 50 ml of 1N aqueous H₂SO₄ solution, the mixture was warmed toRT, and worked up with ether in the usual way giving 3.2 g (90%) ofcrude aldehyde[(+)S-6-[dimethyl(1,1-dimethylethyl)silyl]-2,5,7,8-tetramethyl-chroman-2-carbaldhyde]as a viscous oil which was purified by silica gel chromatography elutingwith 19% (v/v) EtOAc in hexane. Concentration of the solution followedby drying under vacuo for 48 h yielded TBDS aldehyde (78%) as a solid ofmp 66-68° C. ¹H-NMR (CDCl₃/TMS, ppm): 0.12 (s, 6H). 1.1 (s, 9H), 1.38(s, 3H), 1.64 (s, 3H), 2.12 (s, 3H), 2.16 (s, 3H), 2.3-2.2 (m, 2H), 2.53(m, 2H) and 9.82 (d, J=1.4 Hz, 1H); MS (CI, m/z): 349.40 M+H⁺, Calc. forC₂₀H₃₂SiO₃ 348.560.

To a solution of 2.97 g of psedoionol in 10 ml of acetonitrile, therewere added, under stirring and while the temperature was kept below 30°C., 4.53 g of triphenylphosphine hydrochloride which had been obtainedby passing dry hydrogen chloride into a solution of triphenylphosphinein dry ether. After the mixture had then been left standing overnight atroom temperature, the acetonitrile was removed under reduced pressurebelow 50° C. To the residue there were added 4.80 gm of[(+)S-6-[dimethyl(1,1-dimethylethyl)silyl]-2,5,7,8-tetramethylchroman-2-carbaldhyde]in 15 ml of dimethylformamide, and the mixture was stirred. When a clearsolution was obtained, sodium methoxide prepared from 0.352 g of sodiumand 7 ml of anhydrous methanol was stirred in, drop by drop below 15° C.The reaction mixture was turned red by the ylid formed. After theaddition was complete, stirring was continued for 30 min at 10° C.; thenthe mixture was gradually heated to 80° C., when the red colordisappeared. The product was poured into 200 ml of 50% aqueous methanol,dried, and concentrated in vacuo. The residual oil was dissolved in 20ml of ether, and an etheral solution of mercuric chloride was addeduntil no more precipitate formed. When the precipitate was filtered andthe filtrate was washed with water, dried and concentrated, to give 4.7g of yellow oil were obtained. The crude silyl ether mixture of cis andtrans alkene was dissolved in THF and tetra-n-butylammonium fluoride(0.031 mole) was added. After being stirred at 23° C. for 40 minutes,the mixture was poured into water and extracted into ether. The etherextract was dried concentrated and purified by silica gel chromatographyeluting with EtOAc in hexane (1:9) to give2,5,7,8-tetramethyl-2R-(2,6,10-trimethyl-1,3,5,9 E:Zdecatetraen)-6-chromanol (68% yield). ¹H-NMR (CDCl₃/TMS, ppm): 1.28 (s,3H, 2aCH₃), 1.65 (s, 3H), 1.70 (s, 6H) 1.72 (s, 3H), 11.9 (m, 6H), 2.18(s, 3H), 2.35 (S, 6H), 2.53 (t, J=6.6 Hz, 2H, 4CH₂), 5.13-5.27 (m, 3H)and 6.44 (m, 2H); MS (CI, m/z): 395.17 M+H⁺, Calc. for C₂₇H₃₈O₂ 394.60.

A solution of 2,5,7,8-tetramethyl-2R-(2,6,10-trimethyl-1,3,5,9 E:Zdecatetraen)-6-chromanol (0.457 g, 1.16 mmol) in N,N-dimethylformamide(20 mL) was treated with methyl bromoacetate (3.4 g, 8.3 mmol) and anexcess of powdered NaOH (1.2 g, 30 mmol). The resulting yellow slurrywas stirred vigorously for 24 h at room temperature. The reaction wasacidified with 5 N HCl and extracted with diethyl ether (3×30 ml). Thecombined ether layers were washed with H₂O (3×30 ml) and brine (1×30ml), and then dried with Na₂SO₄. The ether solution was concentrated toa yellow oil that was purified by silica gel chromatography eluting with19% (v/v) EtOAc and 2% acetic acid in hexanes. The resulting liquid wasdissolved in diethyl ether (30 ml), washed with H₂O (3×20 mL) and brine(1×20 mL), and then dried with Na₂SO₄. The resulting solution wasconcentrated and dried in vacuo for 48 h. This yielded compound 18 in67% yield. ¹H-NMR (CDCl₃/TMS, ppm): 1.24 (s, 3H, 2aCH₃), 1.63 (s, 3H),1.72 (s, 6H) 1.74 (s, 3H), 1.92 (m, 6H), 2.18 (s, 3H), 2.29 (S, 6H),2.43 (t, J=6.6 Hz, 2H, 4CH₂), 4.68 (s, 2H, OCH₂), 5.10-5.27 (m, 3H) and6.34 (m, 2H); MS (CI, m/z): 452.24 M−H⁺, Calc. for C₂₇H₃₈O₂ 452.63.

3-(2,5,7,8-tetramethyl-(2R-(4R,8,12-trimethyltridecyl)chroman-6-yloxy)propyl-1-ammoniumchloride (19)

A solution of 3-bromopropylamine hydrobromide (1.0 g, 4.6 mmol) in a 2:1dioxane/H₂O (45 mL) was cooled to 0° C. and treated with K₂CO₃ (6.22 g,45 mmol) and di-tert-butyl dicarbonate (1.5 g, 6.9 mmol). The reactionwas stirred for 15 h while warming to room temperature. The dioxane wasremoved in vacuo and the remaining aqueous mixture was acidified with 5N HCl and extracted with ethyl acetate (5×25 mL). The combined organiclayers were dried with MgSO₄ and yielded3-bromo-N-(tert-butoxycarbonyl)propylamine as a colorless oil (0.93 g,93%). ¹H-NMR (CDCl₃/TMS, ppm): 1.41 (s 9H, CH₃), 2.02 (quintet, J=6.4Hz, 2H, CH₂), 3.23 (m, 2H, NCH₂), 3.41 (t, J=6.6 Hz, CH₂Br), 4.8 (broad,1H, NH); ¹³C-NMR (CDCl₃, ppm): 28.3 (CH₃), 30.7, 32.6, 38.9 (CH₂), 79.3(quaternary C), 155.9 (CO); MS (CI, m/z): 239, 241 (M+H⁺ Calc. forC₈H₁₆BrNO₂ 237.03644).

A solution of R,R,R-α-tocopherol (0.5 g, 1.16 mmol) inN,N-dimethylformamide (15 mL) was treated with3-bromo-N-(tert-butoxycarbonyl)propylamine (0.9 g, 3.8 mmol) and anexcess of powdered NaOH (0.32 g, 8 mmol). The resulting yellow slurrywas stirred vigorously for 24 h at room temperature. The reaction wasacidified with 5 N HCl and extracted with diethyl ether (3×30 ml). Thecombined ether layers were washed with H₂O (3×30 ml) and brine (1×30ml), and then dried, with Na₂SO₄. The ether solution was concentrated toa yellow oil that was purified by silica gel chromatography eluting withEtOAc (10% v/v) in hexanes. This yielded desired ether as a colorlessoil (0.45 g, 66%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-,12a′-, 13′-CH₃), 1.0-1.6 (m, 33H, 4′-, 8′-, 12′-CH,1′-, 2′-, 3′-, 5′-,6′-, 7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂), 1.99(quintet, J=6.2 Hz, 2H, CH₂), 2.07, 2.14, 2.16 (3×s, 9H, 5a-, 7a-,8a-CH₃), 2.59 (t, J=6.6 Hz, 2H, 4-CH₂), 3.43 (m, 2H, NCH₂), 3.73 (t,J=5.7 Hz, 2H, OCH₂), 4.34 (s, 2H, OCH₂); ¹³C-NMR (CDCl₃, ppm): 11.7,12.0, 12.9 (5a-, 7a-, 8a-CH₃), 19.6, 19.7 (CH₃), 20.6, 21.0 (CH₂), 22.6,22.7 (CH₃), 23.7 (2a-CH₃), 24.4, 24.8 (CH₂), 27.9 (CH), 31.2 (3-CH₂),32.7, 32.8 (CH), 37.2, 37.4, 37.5, 39.3, 40.1 (CH₂), 70.2 (OCH₂), 74.8(2-C), 117.5, 122.9, 125.5, 127.5 (aryl C), 147.5, 148.0 (aryl C—O),156.0 (CO); MS (CI, m/z): 589 M+H⁺, Calc. for C₃₇H₆₅NO₄ 587.49136.

The above N-protected ether (0.1 g, 0.17 mmol) was dissolved 4 N HCl indioxane (1 mL, 4 mmol) and stirred for 4 h. The dioxane was removed byblowing a stream of argon over the reaction mixture. The resultingmaterial was dried in vacuo for 8 h yielding 19 as a white solid (82 mg,99%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 33H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂), 1.99 (quintet,J=6.2 Hz, 2H, CH₂), 2.07, 2.11, 2.15 (3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.29(m, 2H, CH₂), 2.59 (t, J=6.6 Hz, 2H, 4-CH₂), 3.43 (m, 2H, NCH₂), 3.79(m, 2H, OCH₂) ¹³C-NMR (CDCl₃, ppm): 11.8, 11.9, 12.7 (5a-, 7a-, 8a-CH₃),19.6, 19.7 (CH₃), 20.6, 21.0 (CH₂), 22.6, 22.7 (CH₃), 23.9 (2a-CH₃),24.4, 24.8 (CH₂), 28.0 (CH), 28.4 (CH₃), 31.2 (3-CH₂), 32.7, 32.8 (CH),37.3, 37.4, 37.5, 39.4, 40.0 (CH₂), 74.8 (OCH₂), 75.0 (2-C), 117.5,122.9, 126.0, 127.3 (aryl C), 147.8, 148.0 (aryl C—O); HRMS (CI, m/z):487.438887 (M+H⁺, Calc. for C₃₂H₅₇NO₂ 487.438935).

2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-3-ene-6-yloxy)aceticacid (20)

A solution of R,R,R,-α-tocopherol acetate (2 g, 4.2 mmol) in anhydroustoluene (150 mL) was heated to reflux and then treated with2,3-dichloro-5,6-dicyano-1,4-benzoquinone (0.96 g, 4.2 mmol) in 4portions at 1 h intervals. The reaction was refluxed for 24 h. Duringthis time the reaction mixture became a dark red color and then itprecipitated a light colored solid. The reaction was cooled to roomtemperature, filtered, and the filtrate was concentrated. The resultingdark colored oil was purified by silica gel chromatography eluting withethyl acetate (10%, v/v) in hexanes. This yielded the desired chromeneacetate as a colorless oil (1.74 g, 88%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87(m, 12H, 4a′-, 8a′-, 12a′-, 13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH,1′-, 2′-, 3′-5′-, 6′-, 7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 2.07, 2.13,2.18 (3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.35 (s, 3H, CH₃CO—), 5.61, 6.52 (2×d,J=10.0 Hz, 2H, CH); ¹³C-NMR (CDCl₃, ppm): 11.5, 11.6, 13.1 (5a-, 7a-,8a-CH₃), 14.1 (CH₃), 19.6, 19.7 (CH₃), 20.4, 21.4 (CH₂), 22.6, 22.7(CH₃), 24.4, 24.8 (CH₂), 25.8 (2a-CH₃), 27.9 (CH), 30.8 (3-CH₂), 32.7,32.8 (CH), 37.2, 37.4, 39.4, 41.0 (CH₂), 60.3 (2-C), 117.6, 119.7,122.3, 122.6, 128.9, 129.6 (aryl and vinyl C), 141.2, 148.4 (aryl C—O),169.4 (CO); HRMS (CI, m/z): 471.375799 M+H⁺, Calc. for C₃₁H₅₀O₃470.375996.

A solution of the chromene acetate (1.0 g, 2.13 mmol) in ethanol (20 mL)was treated with 2 N NaOH (20 mL) and stirred at 60° C. for 90 min. Thereaction mixture was cooled, acidified with 5 N HCl, and the ethanol wasremoved in vacuo. The resulting aqueous solution was extracted withether and concentrated to a light yellow oil that was purified by silicagel chromatography eluting with ethyl acetate (15%, v/v) in hexanes.This yielded the desired chromene-6-ol intermediate as a colorless oil(0.92 g, 98%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH, 1′-,2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 2.14, 2.18, 2.19 (3×s, 9H, 5a-, 7a-,8a-CH₃), 5.63, 6.55 (2×d, J=10.0 Hz, 2H, CH); ¹³C-NMR (CDCl₃, ppm):10.8, 11.6, 12.4 (5a-, 7a-, 8a-CH₃), 19.6, 19.7 (CH₃), 21.3 (CH₂), 22.6,22.7 (CH₃), 24.4, 24.8 (CH₂), 25.2 (2a-CH₃), 27.9 (CH), 30.9 (3-CH₂),32.7, 32.8 (CH), 37.2, 37.4, 37.5, 39.3, 40.5 (CH₂), 50.8 (2-C), 116.2,117.8, 120.1, 122.3, 123.0, 130.0 (aryl and vinyl C), 144.6, 145.3 (arylC—O), 169.4 (CO); HRMS (CI, m/z): 428.365275 M+H⁺, Calc. for C₂₉H₄₈O₂428.365431.

A solution of the chromene-6-ol intermediate (0.9 g, 2.1 mmol) inN,N-dimethylformamide (20 mL) was treated with methyl bromoacetate (3.4g, 8.3 mmol) and an excess of powdered NaOH (1.2 g, 30 mmol). Theresulting yellow slurry was stirred vigorously for 24 h at roomtemperature. The reaction was acidified with 5 N HCl and extracted withdiethyl ether (3×30 ml). The combined ether layers were washed with H₂O(3×30 ml) and brine (1×30 ml), and then dried with Na₂SO₄. The ethersolution was concentrated to a yellow oil that was purified by silicagel chromatography eluting with 19% (v/v) EtOAc and 2% acetic acid inhexanes. The resulting yellow liquid was dissolved in diethyl ether (30ml), washed with H₂O (3×20 mL) and brine (1×20 mL), and then dried withNa₂SO₄. The resulting solution was concentrated to a light yellow oiland dried in vacuo for 48 h. This yielded 19 as a colorless (0.90 g,88%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 2.07, 2.10, 2.19 (3×s, 9H, 5a-, 7a-,8a-CH₃), 4.37 (s, 2H, OCH₂), 5.62, 6.50 (2×d, J=10.0 Hz, 2H, CH);¹³C-NMR (CDCl₃, ppm): 11.3, 11.5, 12.9 (5a-, 7a-, 8a-CH₃), 19.6, 19.7(CH₃), 21.3 (CH₂), 22.6, 22.7 (CH₃), 24.4, 24.8 (CH₂), 25.6 (2a-CH₃),27.9 (CH), 30.9 (3-CH₂), 32.7, 32.8 (CH), 37.2, 37.4, 37.5, 39.3, 40.9(CH₂), 60.5 (OCH₂), 69.1 (2-C), 118.0, 119.8, 122.8, 122.9, 129.6, 19.8(aryl and vinyl C), 147.5, 147.8 (aryl C—O), 173.4 (CO); HRMS (CI, m/z):487.378731 M+H⁺, Calc. for C₃₁H₅₁O₄ 487.378736.

2-(2,5,7,8-tetramethyl-(2R-(4R,8,12-trimethyltridecyl)chroman-6-yloxy)triethylammoniumsulfate (21)

A solution of2-(2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy))ethan-1-ol(13) (0.1 g, 0.21 mmol) in anhydrous DMF (2 mL) and pyridine (0.6 mL)was treated sulfur trioxide-N,N-dimethylformamide complex (0.16 g, 1.0mmol), and the resulting solution was stirred for 24 h. The reactionmixture was quenched with 1 N HCl and then extracted with CH₂Cl₂ (5×5mL). Gaseous ammonia was bubbled through the CH₂Cl₂ solution for 10 min.The resulting solution was concentrated to a yellow paste and purifiedby silica gel chromatography eluting with MeOH (10%, v/v) and triethylamine (2%) in CHCl₃. This yielded 21 as a yellow semi-solid (92 mg, 77%)¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-, 13′-CH₃),1.0-1.6 (m, 33H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-, 7′-, 9′-,10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂), 1.95 2.01, 2.05 (3×s, 9H,5a-, 7a-, 8a-CH₃), 2.45 (t, J=6.6 Hz, 2H, 4-CH₂), 3.05 (m, 6H, NCH₂),3.79 (m, 2H, OCH₂), 4.21 (m, 2H, OCH₂); ¹³C-NMR (CDCl₃, ppm): 9.46(CH₃), 12.4, 12.6, 13.5 (5a-, 7a-, 8a-CH₃), 20.3, 20.4 (CH₃), 21.3, 21.7(CH₂), 23.3, 23.4 (CH₃), 24.5 (2a-CH₃), 25.1, 25.5 (CH₂), 28.6 (CH),31.9 (3-CH₂), 33.3, 33.4 (CH), 37.9, 38.1, 40.0, 40.8 (CH₂), 46.9(NCH₂), 67.4, 71.9 (OCH₂), 75.5 (2-C), 118.3, 123.5, 126.5, 128.3 (arylC), 148.5 (aryl C—O); HRMS (CI, m/z): 554.364102 M-NH₃, Calc. forC₃₁H₅₄O₆S 554.364119.

6-(2,5,7,8-tetramethyl-(2R-(4R,8,12-trimethyltridecyl)chroman)aceticacid (22)

A solution of R,R,R-α-tocopherol (1.0 g, 2.3 mmol) in anhydrous CH₂Cl₂(25 mL) was cooled to 0° C. Diisopropylethyl amine (2 mL, 11.6 mmol) wasadded followed by the dropwise addition of trifluoromethylsulfonicanhydride (5.0 g, 17.7 mmol). The solution turned to a dark immediatelyand was allowed to warm to room temperature while stirring for 24 h. Thereaction was quenched with H₂O and then was extracted with diethyl ether(2×100 mL). The combined ether layers were washed with 1 N HCl (50 mL),H₂O (50 mL), brine (50 mL), and then dried with MgSO₄. The ethersolution was concentrated to a yellow oil and purified by silica gelchromatography eluting with ethyl acetate (3%, v/v) in hexane. Thisyielded the desired triflate intermediate as a yellow oil (1.3 g,quantitative). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂), 2.07, 2.13, 2.21(3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.62 (t, J=6.6 Hz, 2H, 4-CH₂); ¹³C-NMR(CDCl₃, ppm): 11.9, 13.2, 14.0 (5a-, 7a-, 8a-CH₃), 19.6, 19.7 (CH₃),20.6, 21.0 (CH₂), 22.6, 22.7 (CH₃), 23.8 (2a-CH₃), 24.4, 24.8 (CH₂),28.0 (CH), 31.2 (3-CH₂), 32.7, 32.8 (CH), 37.3, 37.4, 37.5, 39.4, 40.0(CH₂), 75.6 (2-C), 118.4, 124.4, 126.7, 128.1 (aryl C), 139.6, 150.9(aryl C—O); ¹⁹F-NMR (CDCl₃, ppm): −73.52; HRMS (CI, m/z): 563.337803(M+H⁺, Calc. for C₃₀H₅₀O₄F₃S 563.338192).

A solution of the triflate (1.3 g, 2.31 mmol) in anhydrous DMF (23 mL)was treated with LiCl (0.98 g, 4.62 mmol), triphenylphosphine (0.37 g,1.4 mmol), 2,6-di-tert-butyl-4-methylphenol (2-3 crystals),tributyl(vinyl)tin (0.73 g, 2.31 mmol), anddichlorobis(triphenylphosphine)-palladium(II) (0.24 g, 0.35 mmol). Thismixture was heated to 120° C. and stirred. After 2 h, additionaltributyl(vinyl)tin (0.73 g, 2.31 mmol). After 8 h, the reaction wascooled to room temperature and added to a mixture of H₂O (50 mL) anddiethyl ether (50 mL). The ether layer was washed with 1 N HCl (6×30 mL)and a saturated solution of KF (6×30 mL). The ether solution was driedwith Na₂SO₄ and then concentrated to a dark oil. This material waspurified by silica gel chromatography eluting with ethyl acetate (3%,v/v) in hexane yielding the 6-vinylchroman intermediate as a clear oil(0.38 g, 38%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H, 4a′-, 8a′-, 12a′-,13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH, 1′-, 2′-, 3′-, 5′-, 6′-,7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.86 (m, 2H, 3-CH₂), 2.20, 2.24, 2.28(3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.62 (t, J=6.8 Hz, 2H, 4-CH₂), 5.18, 5.56(2×dd, J_(gem), =2.3 Hz, J_(cis)=11.2 Hz, J_(trans)=18.7 Hz, 2H, ═CH₂),6.77 (dd, J=18.7, 11.2 Hz, 1H, CH); ¹³C-NMR (CDCl₃, ppm): 11.9, 16.3,17.2 (5a-, 7a-, 8a-CH₃), 19.7, 19.8 (CH₃), 20.8, 21.1 (CH₂), 22.6, 22.7(CH₃), 23.9 (2a-CH₃), 24.5, 24.8 (CH₂), 28.0 (CH), 31.2 (3-CH₂), 32.7,32.8 (CH), 37.3, 37.5, 37.5, 39.4, 40.1 (CH₂), 74.9 (2-C), 116.7, 119.0,122.0, 129.8, 131.2, 132.8, 136.8 (aryl/vinyl C), 150.9 (aryl C—O); HRMS(CI, m/z): 440.401602 (M+H⁺, Calc. for C₃₁H₅₂O 440.401812).

A solution of the 6-vinylchroman intermediate (0.12 g, 0.27 mmol) inanhydrous THF (1 mL) was cooled to 0° C. and treated with 9-BBN (0.60mL, 0.5 M in THF, 0.3 mmol). The reaction mixture was heated to refluxfor 8 h. The reaction was quenched with water (1.5 mL) and treated withNaBO₃.4H₂O and the resulting slurry was stirred overnight. Diethyl ether(4 mL) and the reaction mixture were extracted with CH₂Cl₂ (2×20 mL).The organic layers were concentrated to a clear oil that was purified bysilica gel chromatography eluting with ethyl acetate (50%, v/v) inhexane. This yielded the desired 6-(2-hydroxyethyl)chroman intermediateas a colorless oil (30 mg, 24%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H,4a′-, 8a′-, 12a′-, 13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′-, 12′-CH, 1′-,2′-, 3′-, 5′-, 6′-, 7′-, 9′-, 10′-, 2a-CH₃), 1.81 (m, 2H, 3-CH₂), 2.17,2.24, 2.28 (3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.68 (t, J=6.8 Hz, 2H, 4-CH₂),3.01 (t, J=7.5 Hz, 2H, Ar—CH₂), 3.74 (t, J=7.5 Hz, 2H, OCH₂); ¹³C-NMR(CDCl₃, ppm): 12.0, 15.1, 16.0 (5a-, 7a-, 8a-CH₃), 19.6, 19.7 (CH₃),20.6, 21.0 (CH₂), 22.6, 22.7 (CH₃), 23.8 (2a-CH₃), 24.4, 24.8 (CH₂),28.0 (CH), 31.2 (3-CH₂), 32.7, 32.8 (CH), 37.3, 37.4, 37.5, 39.4, 40.0(CH₂), 62.2 (OCH₂), 72.6 (2-C), 116.8, 122.3, 124.9, 132.4, 133.9 (arylC), 150.1 (aryl C—O); HRMS (CI, m/z): 458.412154 (M+H⁺, Calc. forC₃₁H₅₄O₂ 458.412384).

A solution of pyridinium chlorochromate (32 mg, 0.1 mmol) in anhydrousCH₂Cl₂ (0.5 mL) was treated with a solution of the6-(2-hydroxyethyl)chroman intermediate (32 mg, 0.07 mmol) in CH₂Cl₂ (0.5mL). The reaction was stirred for 2 h at which time n o startingmaterial was visible by thin layer chromatography. Diethyl ether (2 mL)was added and the resulting solution was filtered through a thin pad ofcelite. The filtrate as concentrated and yielded a yellow oil (20 mg).This oil was dissolved in t-BuOH (0.5 mL) and treated with phosphatebuffer (0.5 mL, 1 N, pH=4.0), 2-methyl-2-butene (0.1 mL) and NaClO₂ (5.4mg, 0.05 mmol). After stirring for 40 min, the reaction mixture wasextracted with CHCl₃ (6×10 mL) and the combined organic layers weredried with Na₂SO₄. The CHCl₃ solution was concentrated to a yellow oilthat was purified by preparative thin layer chromatography eluting withethyl acetate (30%, v/v) and acetic acid (1%) in hexanes. This yielded22 as colorless oil (20 mg, 63%). ¹H-NMR (CDCl₃/TMS, ppm): 0.87 (m, 12H,4a′-, 8a′-, 12a′-, 13′-CH₃), 1.0-1.6 (m, 24H, 4′-, 8′, 12′-CH, 1′-, 2′-,3′-, 5′-, 6′-, 7′-, 9′-, 10′-, 11′-CH₂, 2a-CH₃), 1.81 (m, 2H, 3-CH₂),2.17, 2.24, 2.28 (3×s, 9H, 5a-, 7a-, 8a-CH₃), 2.66 (t, J=6.8 Hz, 2H,4-CH₂), 3.71 (s, 2H, CH₂COOH); ¹³C-NMR (CDCl₃, ppm): 12.0, 15.3, 16.2(5a-, 7a-, 8a-CH₃), 19.6, 19.7 (CH₃), 20.6, 21.0 (CH₂), 22.6, 22.7(CH₃), 23.8 (2a-CH₃); 24.4, 24.8 (CH₂), 28.0 (CH), 28.9, 31.2 (3-CH₂),32.7, 32.8 (CH), 37.3, 37.4, 37.5, 39.4, 40.0 (CH₂), 72.6 (2-C), 117.1,122.2, 124.9, 132.4, 132.7 (aryl C), 150.2 (aryl C—O), 179.2 (COOH);HRMS (CI, m/z): 472.391583 (M+H⁺, Calc. for C₃₁H₅₂O₃ 472.391644).

2,5,7,8-tetramethyl-(2R-(heptyl)chroman-6-yloxy)acetic acid (23)

A solution of hexyltriphenyphosphonium bromide (0.880 g, 2.05 mmol) in11.2 ml of anhydrous DME was stirred at room temperature while 0.86 ml(2.06 mmol) of 2.4 M n-butyllithium in hexane was added. The resultingred solution was stirred for 2 h at room temperature, then a solution of[(+)S-6-Benzyloxy-2,5,7,8-tetramethylchroman-2-carbaldhyde (306 mg,0.944 mmol) in 3 ml of anhydrous DME was added and stirring wascontinued for 3 h at 65-75° C. After cooling, the reaction mixture waspoured into cold dilute H₂SO₄ and work up ether was carried out in theusual manner. The ether was concentrated in vacuo to afford the oilymaterial. Product was isolated using column chromatography and elutedwith chloroform to yield 46% of the product. The mixture of cis andtrans alkene was dissolved in 30 ml of ethyl acetate and 50 mg of 5%palladium on carbon was added, and the mixture was shaken under 40 psiof H₂ for 10 hrs and then filtered through Celilte and rinsed well withethyl acetate. The filtrate was concentrated and purified by silica gelchromatography eluting with EtOAc in hexane (1:9) to give (2R)2,5,7,8-tetramethyl-2-(heptyl)-6-chromanol (60% yield) ¹H-NMR(CDCl₃/TMS, ppm): 0.89 (s, 3H), 1.3-1.5 (m, 15H), 1.89 (m, 2H), 2.2 (s,3H), 2.08 (s, 3H), 2.23 (s, 3H), and 2.48 (t, J=6.5 Hz, 2H); MS (CI,m/z):305.35 M+H⁺, Calc. for C₂₀H₃₂O₂ 304.4746).

A solution of 2,5,7,8-tetramethyl-2-(heptyl)chromanol (0.353 g, 1.16mmol) in N,N-dimethylformamide (20 mL) was treated with methylbromoacetate (3.4 g, 8.3 mmol) and an excess of powdered NaOH (1.2 g, 30mmol). The resulting yellow slurry was stirred vigorously for 24 h atroom temperature. The reaction was acidified with 5 N HCl and extractedwith diethyl ether (3×30 ml).

The combined ether layers were washed with H₂O (3×30 ml) and brine (1×30ml), and then dried with Na₂SO₄. The ether solution was concentrated toa yellow oil that was purified by silica gel chromatography eluting with19% (v/v) EtOAc and 2% acetic acid in hexanes. The resulting liquid wasdissolved in diethyl ether (30 ml), washed with H₂O (3×20 mL) and brine(1×20 mL), and then dried with Na₂SO₄. The resulting solution wasconcentrated and dried in vacuo for 48 h. This yielded compound 23 in36% yield. (CDCl₃/TMS, ppm): ¹H-NMR (CDCl₃/TMS, ppm): 0.88 (s, 3H),1.2-1.5 (m, 15H), 1.88 (m, 2H), 2.1 (s, 3H), 2.18 (s, 3H), 2.2 (s, 3H),2.55 (t, J=6.5 Hz, 2H) and 4.78 (s, 2H); HRMS (CI, m/z): 363.2535 (M+H⁺,Calc. for C₂₂H₃₅O₄363.2541).

2,5,7,8-tetramethyl-(2R-(tridecyl)chroman-6-yloxy)acetic acid (24)

The compounds 24 and 25 were synthesized in manner identical to thesynthesis of 23 using appropriate phosphonium bromide.

¹H-NMR (CDCl₃/TMS, ppm): 0.83 (s, 3H), 1.25-1.57 (m, 27H), 1.88 (m, 2H),2.1 (s, 3H), 2.18 (s, 3H), 2.20 (s, 3H), 2.55 (t, J=6.6 Hz, 2H) and 4.48(s, 2H); MS (CI, m/z): 447.14 M+H⁺, Calc. For C₂₈H₄₆O₄ 446.6732.

2,5,7,8-tetramethyl-(2R-(heptadecyl)chroman-6-yloxy)acetic acid (25)

¹H-NMR (CDCl₃/TMS, ppm): 0.86 (s, 3H), 1.15-1.67 (m, 35H), 1.88 (m, 2H),2.16 (s, 3H), 2.20 (s, 3H), 2.23 (s, 3H), 2.55 (t, J=6.4 Hz, 2H) and4.78 (s, 2H); MS (CI, m/z): 503.45 M+H⁺, Calc. For C₃₂H₅₄O₄ 502.781.

2,5,7,8-tetramethyl-2R-(4,8,-dimethyl-1,3,7 E:Znonotrien)chroman-6-yloxy)acetic acid (26)

Compound 26 was synthesized in a manner identical to the synthesis ofcompound 18 using nerol instead of pseudoionol. ¹H-NMR (CDCl₃/TMS, ppm):1.24 (s, 3H, 2aCH₃), 1.63 (m, 1H), 1.68 (s, 3H), 1.74 (s, 6H), 1.92 (m,6H), 2.18 (s, 3H), 2.29 (S, 6H), 2.43 (t, J=6.6 Hz, 2H, 4CH₂), 4.68 (s,2H, OCH₂), 5.64 (m, 2H) and 5.27 (m, 1H); MS (CI, m/z): 413.24 M+H⁺,Calc. for C₂₆H₃₆O₄412.0115.

E.Z,RS,RS,RS-(phytyltrimethylbenzenethiol-6-yloxy)acetic acid (27)

2,3,6-trimethylphenol (1.6 g, 11.8 mmol) was dissolved in 50 mL ofanhydrous methanol which had been deoxygenated by bubbling withnitrogen. Ammonium thiocyanate (2.2 g, 28.9 mmol) was added to thissolution which was then cooled to 0° C. and bubbled with chlorine gas.The initially colorless homogeneous solution becomes pink and then greenwith the formation of a white precipitate. The solution was stirred for1 h at 0° C. and then for a further hour at 20° C. The dissolvedchlorine was removed by bubbling with nitrogen and the precipitateremoved by filtration. Evaporation of the filtrate under reducedpressure followed by drying under high vacuum (0.1 torr) yielded 2.20 g(97%) of 2,3,5-Trimethyl-4-hydroxyphenylthiocyanate in a form pureenough for the next step in the synthesis. An analytical sample wasrecrystallized from hexanes: white crystals, mp 100.3° C. ¹H NMR (CDCl₃)δ 7.2 (s, 1H), 5.0 (s, 1H) 2.4 (s, 3H), 2.2 (s, 6H).

2,3,5-Trimethyl-4-hydroxyphenylthiocyanate (2 g, 10.35 mmol) wasdissolved in 100 mL of anhydrous ether containing 25 mL of anhydroustetrahydrofuran. This solution was added dropwise over 1 h to 100 mL ofanhydrous ether containing LiAlH₄ (0.9 g, 24 mmol) at room temperature.After a further hour at 20° C., the unreacted LiAlH₄ was destroyed bycooling the heterogeneous mixture to 0° C. and adding moist ether (50mL), H₂O (50 mL), and 1 N HCl (50 mL). A further 50 mL of water wasadded and the organic phase was separated and washed with water (2×50mL), NaHCO₃ solution (2×50 mL), water (2×50 mL), and saturated NaCl (50mL). The organic phase was dried over anhydrous MgSO₄ and filtered andthe solvent removed under reduced pressure. Silica gel columnchromatography with 5% ethyl acetate in hexane gave 1.8 g (90%) of2,3,5-trimethyl-4-hydroxybenzenethiol as a white powder, mp 86° C.[Lit.¹ mp 86° C.].

Solution of 2,3,5-trimethyl-4-hydroxybenzenethiol (3 g, 17.83 mmol),isophytol (4.8 g, 16.19 mmol), anhydrous zinc chloride (1.2 g, 8.8 mmol)and 0.2 mL of glacial acetic acid in 30 mL of absolute ether wasrefluxed for 1 h. The solvent was then removed in vacuo at 50° C. andthe red oil obtained was dissolved in a mixture of 50 mL of petroleunether and 20 mL of 70% aqueous methanol. The ether layer was dried(Na₂SO₄) and evaporated in vacuo to give a red oil, which was purifiedby silica gel chromatography eluting with hexans:ether (9:1) to give 3 g(38%) E.Z, RS, RS, RS-Phytyltrimethylhydroxybenzenethiol as yellow oil.¹H NMR (CDCl₃) δ7.11 (s, 1H, Ar—H), 5.23 (t, 1H, vinylic-H), 4.62 (s,1H, OH), 3.34 (d, 2H, Ar—S—CH₂—), 2.41 (s, 3H, Ar—CH₃), 2.19 (s, 3H,Ar—CH₃), 2.18 (s, 3H, Ar—CH₃), 0.83-1.92 (m, 39H, Phytol chain).

A solution of phytyltrimethylhydroxybenzenethiol (3 g, 6.7 mmol) inN,N-dimethyl-formamide (80 mL) was treated with methyl bromoacetate (7.4g, 48.3 mmol) and an excess of powdered NaOH (7 g, 175 mmol). Theresulting pink oil was stirred at RT for 24 h. The reaction mixture wasacidified with 5 N HCl and extracted with ether (3×150 mL). The combinedether layers were washed with H₂O (3×150 mL) and brine (1×150 mL), andthen dried (Na₂SO₄). The ether solution was concentrated to a yellow oilthat was purified by silica gel chromatography eluting with 20% EtOAc inhexane to give 3 g (88%) of E.Z, RS, RS,RS-(phytyltrimethylbenzenethiol-6-yloxy)acetic acid as a yellow oil. ¹HNMR (CDCl₃) δ 10.90 (s, 1H, COOH), 8.08 (s, 1H, Ar—H), 5.30 (t, 1H,vinylic-H), 4.35 (s, 2H, CH₂COOH), 3.42 (d, 2H, Ar—S—CH₂—), 2.34 (s, 3H,Ar—CH₃), 2.25 (s, 3H, Ar—CH₃), 2.22 (s, 3H, Ar—CH₃), 0.83-1.94 (m, 39H,Phytyl chain). HRMS (CI, m/z): 504.362821 (M+H⁺, Calc. for C₃₁H₅₃O₃S504.363718).

(R)-2[(2,5,7,8-tetramethyl-2-(3 propene methyl ester)chroman-6-yloxy]acetic acid (28)

To a slurry of (carbomethoxymethyl)triphenyl phosphonium bromide (1.8gm, 4.32 mmol) in 12 ml of THF at ° C. was added 1.66 ml of n-BuLi (2.5Min hexane) dropwise. The resulting solution was removed to roomtemperature for 2 h, and then a solution of(+)S-6-[dimethyl(1,1-dimethylethyl)silyl]-2,5,7,8-tetramethylchroman-2-carbaldhyde (1.31 g, 3.76 mmol) in 7 ml THF was added viacannula. The solution was stirred at room temperature for 44 hr and then10 ml of 1N aq. HCl was added. The layer were separated and then aq.phase was extracted with ether (3×15 ml). The combined organic layerwere washed with brine, dried over Na₂SO₄ and filtered. Afterconcentration of the filtrate, the crude alkene was purified by flashchromatography eluting with dichloromethane to give mixture of the cisand trans alkene in 93% yield. The silyl ether mixture of cis and transalkene (3.76 mmol) was dissolved in THF andtetra-n-butylammoniumfluoride (0.041 mole) was added. After beingstirred at 23° C. for 1.5 h, the mixture was poured into water andextracted into ether. The ether extract was dried concentrated andpurified by silica gel chromatography eluting with EtOAc in hexane (3:7)and both the cis and trans isomer of2,5,7,8-tetramethyl-2R-(3′propenemethyl ester)-6-chromanol were isolatedand characterized (68% yield) ¹H-NMR (CDCl₃/TMS, ppm): 1.65 (s, 3H, 2aCH₃), 2.12 (m, 2H, 3CH₂), 2.39 (s, 9H, CH₃), 2.48 (m, 2H, 4 CH₂), 3.78(s, 3H, OCH₃), 6.11 (d, 1H, CH═) and 7.13 (d, 1H, CH═).

A solution of 2,5,7,8-tetramethyl-2R-(3′propene methylester)-6-chromanol (0.353 g, 1.16 mmol) in N,N-dimethylformamide (20 mL)was treated with methyl bromoacetate (3.4 g, 8.3 mmol) and an excess ofpowdered NaOH (1.2 g, 30 mmol). The resulting yellow slurry was stirredvigorously for 24 h at room temperature. The reaction was acidified with5 N HCl and extracted with diethyl ether (3×30 ml). The combined etherlayers were washed with H₂O (3×30 ml) and brine (1×30 ml), and thendried with Na₂SO₄. The ether solution was concentrated to a yellow oilthat was purified by silica gel chromatography eluting with 19% (v/v)EtOAc and 2% acetic acid in hexanes. The resulting liquid was dissolvedin diethyl ether (30 ml), washed with H₂O (3×20 mL) and brine (1×20 mL),and then dried with Na₂SO₄. The resulting solution was concentrated anddried in vacuo for 48 h. This yielded compound 28 in 40% yield. ¹H-NMR(CDCl₃/TMS, ppm): 1.68 (s, 3H, 2a CH₃), 2.11 (m, 2H, 3CH₂), 2.36 (s, 9H,CH₃), 2.56 (m, 2H, 4 CH₂), 3.70 (s, 31-1, OCH₃), 4.78 (s, 2H, OCH₂),6.03 (d, 1H, CH═) and 7.03 (d, 1H, CH═); MS (CI, m/z):337.24 M+H⁺, Calc.for C₁₈H₂₄O₆336.3867.

2,5,7,8-tetramethyl-(2R-(methyl propionate)chroman-6-yloxy)acetic acid(29)

The mixture of cis and trans alkene 2,5,7,8-tetramethyl-2R-(3 propenemethylester)-6-chromanol was dissolved in 30 ml of ethyl acetate and 50mg of 5% palladium on carbon was added, and the mixture was shaken under40 psi of H₂ for 24 hrs and then filtered through Celilte and rinsedwell with ethyl acetate. The filtrate was concentrated and purified bysilica gel chromatography eluting with EtOAc in hexane (1:9) to givecompound #29. ¹H-NMR (CDCl₃/TMS, ppm): 1.62 (s, 3H, 2a CH₃), 2.0-2.3 (m,6H, CH₂), 2.41 (s, 9H, CH₃), 2.53 (m, 2H, 4 CH₂), 3.67 (s, 3H, OCH₃) and4.88 (s, 2H, OCH₂); MS (CI, m/z): 339.34 M+H⁺, Calc. forC₁₈H₂₆O₆338.4025.

EXAMPLE 3 Synthesis of all-racemic 1-aza-α-tocopherol analogsPhytyltrimethylhydroquinone (31)

The solution of trimethylhydroquinone (30, 7.5 g, 49.28 mmol), isophytol(12 g, 40.47 mmol), anhydrous zinc chloride (3 g, 22.01 mmol) and 0.4 mLof glacial acetic acid in 60 mL of absolute ether was refluxed for 1 h.The solvent was then removed in vacuo at 50° C. and the slurry obtainedwas dissolved in a mixture of 50 mL of petroleum ether and 20 mL of 70%aqueous methanol. The emulsion formed was destroyed by the addition of20 mL of ether. The ether layer was dried (Na₂SO₄) and evaporated invacuo to give a red solid paste that was triturated vigorously with 70mL of petroleum ether. After cooling to −78° C., the suspension wascentrifuged and the supernatant petroleum ether was decanted. Coldpetroleum ether (10 mL) was added to the crystalline mass, and theoperation was twice repeated to give 10 g (47%) of sticky white solid,which was used in the next step without further purification.

Phytyltrimethylhydroquinone diacetate (32)

The crude phytyltrimethylhydroquinone (31, 9 g, 20.1 mmol) was dissolvedin dry pyridine (90 g) and acetic anhydride (90 g). After storage for 10h at room temperature, the mixture was poured onto ice (200 mL) andextracted with ether (2×100 mL). The ether solution was washed with 3Nsulfuric acid (2×100 mL), 10% sodium bicarbonate solution (2×100 mL),and again water (2×100 mL), dried (Na₂SO₄), and evaporated to yield 10.2g (95%) of diacetate (32) as a light yellow oil. The oil was used in thenext step without further purification.

Trimethyl(3,7,11,15-tetramethyl-3-benzamidohexadecyl)hydro quinonediacetate (33)

To a mixture of benzonitrile (10 g, 96.97 mmol), concentrated sulfuricacid (10 g) and glacial acetic acid (40 mL) was added, with stirring at0° C., the diacetate (32, 10 g, 19.43 mmol). The solution was stored for10 h at room temperature, then poured onto ice (150 mL) and extractedwith ether (3×100 mL). The ether extracts were washed neutral with water(3×100 mL), and saturated sodium bicarbonate solution, dried (Na₂SO₄)and evaporated in vacuo. The crude product (21 g) was chromatographed onsilica gel eluting with petroleum ether:ether (9:1). The unchangedbenzonitrile was first removed. Elution with ether then afforded 11 g(88%) of product which was crystallized from petroleum ether to giveanalytical sample (33) m.p. 90-92° C. (lit.194-95° C.).

2,5,7,8-Tetramethyl-2(4,8,12-trimethyltridecyl)3,4-dihydroquinolin-6(2H)-one(37)

To a solution of the diacetate (33, 13 g, 20.4 mmol) in 60 mL ofmethanol, solid potassium hydroxide (11 g) was added and the mixture wasrefluxed under argon for 45 min. After cooling, the solution was dilutedwith ice-water (100 mL), and extracted under carbon dioxide with ether(3×100 mL) yielding 10 g (88%) of crude hydroquinone (34).

A solution of this product (34, 10 g, 18.1 mmol) in absolute ether (110mL) was added dropwise with stirring during 20 min. to a suspension oflithium aluminum hydride (20 g, 527 mmol) in absolute ether (110 mL).The mixture was refluxed for 14 h and the n hydrolyzed under carbondioxide by the dropwise addition of methanol (10 mL) followed by 3Nhydrochloric acid (30 mL). After extraction with ether (3×200 mL), thecrude solid benzylamino derivative (35, 8.4 g, 86%) was obtained.

This material (35, 8.4 g, 15.6 mmol) was dissolved in glacial aceticacid (120 mL) and hydrogenated at 50° C. and atmospheric pressure in thepresence of 2 g of palladium on charcoal (5%) at room temperature for 22h (or until the hydrogen absorption ceased). After cooling, the catalystwas filtered off, and the solution was diluted with water (50 mL),extracted with ether (3×80 mL) and dried (Na₂SO₄).

To this dry and neutral ether solution of 36 was added fresh silveroxide (2.1 g, 8.93 mmol), and the suspension was stirred for 14 h. Afterfiltration under argon and evaporation, 4 g of the crude iminoquinone(37) was obtained, which was chromatographed on silica gel eluting withhexanes:ether (200:1) to yield 2.4 g (63%) of pure yellow oil. HMRS (cl,m/z): 428.389883 (M+H⁺, Calc. For C₂₉H₄₉NO 428.389241).

1-Aza-α-tocopherol (38)

The iminoquinone (37, 1.34 g, 3.133 mmol) in 10 mL of ether washydrorgenated at room temperature and atmospheric pressure in thepresence of 190 mg of Lindlar's catalyst. After 20 h, the catalyst wasfiltered off under argon and the filtrate was evaporated in vacuo togive 810 mg (60%) of crude red oil, which was used in the next stepwithout further purification.

1-Aza-α-tocopherol-6-yloxyl-acetic acid (39) and1-Aza-α-tocopherol-6-yloxyl-methyl acetate (40)

A solution of 1-Aza-α-tocopherol (38, 800 mg, 1.86 mmol) in DMF (30 mL)was treated with methyl bromoacetate (2.1 g, 13.4 mmol) and an excess ofpowdered NaOH (1.9 g, 48.4 mmol). The resulting yellow suspension wasstirred vigorously for 24 h at room temperature. The reaction mixturewas acidified with 5N HCl to pH6, and extracted with diethyl ether (3×30mL). The combined ether layers were washed with water (3×40 mL), brine(1×40 mL), and then dried (Na2SO4). The ether solution was concentratedto a yellow oil that was purified by silica gel chromatography elutingwith CH₂Cl₂:MeOH:Hac (97:2:1) to give 300 mg (33%) of sticky yellowsolid (39). ¹H-NMR (CDCl₃, ppm) 4.30 (s, 2H, OCH₂), 3.30 (s, 1H, NH),2.59 (t, 2H, Ar—CH₂), 2.18 (s, 3H, Ar—CH₃), 2.12 (s, 3H, Ar—CH₃), 2.00(s, 3H, Ar—CH₃), 1.80-0.84 (m, 38H, Ar—CH₂CH₂, NCCH₃ and phytyl chain);¹³C-NMR (CDCl₃, ppm): 12.01, 12.77, 12.96, 19.60, 19.67, 19.72, 21.09,22.60, 22.71, 24.42, 24.79, 26.56, 27.95, 32.11, 32.74, 37.26, 37.37,37.42, 37.55, 37.65 39.34, 41.36, 50.92, 69.50 (aliphitic C), 117.69,118.55, 125.99, 126.39, 138.07, 146.05 (Ar—C), 173.18 (COOH); (HRMS (CI,m/z): 488.411055 (M+H⁺, Calc for C₃₁H₅₃NO₃ 488.410370) and 150 mg (17%)of yellow oil (40). (CDCl₃/TMS, ppm) 4.31 (s, 2H, OCH₂), 3.85 (S, 3H,CO₂CH₃), 3.32 (S, 1H, NH), 2.61 (T, 2H, Ar—CH₂), 2.23 (S, 3H, Ar—CH₃),2.17 (S, 3H, Ar—CH₃), 2.01 (S, 3H, Ar—CH₃), 1.81-0.86 (M, 38H,Ar—CH₂CH₂, NCCH₃ and phytyl chain); ¹³C-NMR (CDCl₃, ppm) 11.96, 12.67,12.90, 19.57, 19.63, 19.69, 21.06, 21.95, 22.57, 22.68, 24.38, 24.74,26.49, 27.91, 32.25, 32.67, 32.70, 37.21, 37.33, 37.53, 39.31, 41.68,50.43, 51.87, 69.97, (aliphitic C), 116.98, 117.72, 126.01, 126.36,138.43, 146.18 (Aryl C), 169.83 (C═O); HRMS (CI, m/z): 502.425330 (MCalc. for C32H55NO3 502.426020).

1-Aza-N-methyl-α-tocopherol-6-yloxyl-methyl acetate (41)

Following the literature procedure 2, a mixture of powdered KOH (500 mg,7.77 mmol) in DMSO (30 mL) was stirred at 0° C. for 5 min. The methylacetate (40, 1.3 g, 23.32 mmol) was added, followed immediately by theaddition of CH₃I (3.3 g, 23.32 mmol). Stirring was continued for 0.5 h(0° C.) after which the mixture was poured into water. Extracts (CH2Cl2,3×40 mL) of the final mixture were combined, washed with water (40 mL),brine (40 mL), dried (MgSO₄) and then evaporated. The resulting yellowoil was chromatographed over silica gel eluting with 5% ETOAC in hexaneto give 810 mg (60%) of light yellow oil. ¹H-NMR (CDCl₃/TMS, ppm) 4.34(s, 2H, OCH₃), 3.84 (s, 3H, CO₂CH₃), 2.55 (t, 2H, Ar—CH₂), 2.49 (s, 3H,NCH₃), 2.24 (s, 3H, Ar—CH₃), 2.20 (s, 3H, Ar—CH₃), 2.13 (s, 3H, Ar—CH₃),1.82-0.82 (m, 38H, Ar—CH₂CH₂, NCCH₃ and phytyl chain); ¹³C-NMR (CDCl₃,ppm) 11.72, 13.12, 14.27, 19.55, 19.68, 20.93, 22.57, 22.66, 22.88,24.35, 24.74, 25.33, 25.37, 27.90, 32.59, 32.71, 37.23, 37.33, 37.51,37.63, 38.49, 39.06, 39.29, 51.14, 69.57 (aliphitic C), 125.34, 126.36,127.42, 130.32, 143.89, 150.33 (Ar—C), 169.73 (C═O). The compound 41 wasused in the next step without further purification.

1-Aza-N-methyl-α-tocopherol-6-yloxyl-acetic acid (42)

To the mixture of the ester (41, 740 mg, 14.34 mmol) in ethanol (34 mL)was added dropwise 2N NaOH (9 mL, 10 eq. 14.34 mmol). After stirring thelight yellow solution at room temperature for 4 h, the reaction mixturewas acidified with 2N HCl to pH6, and then extracted with EtOAc (3×100mL) and the combined organic layers were washed with water (2×100 mL),brine (1×100 mL), dried (MgSO4), and evaporated to give a light yellowoil. The oil was chromatographed over silica gel eluting with 1% HOAc,30% EtOAc in hexans to give 650 mg (90%) of the product (42) as acolorless oil. ¹H-NMR (CDCl₃/TMS, ppm) 10.43 (s, 1H, CO₂H), 4.37 (s, 1H,OCH₂), 2.51 (t, 2H, Ar—CH₂), 2.43 (s, 3H, NCH₃), 2.22 (Ar—CH₃), 2.12 (s,3H, Ar—CH₃), 1.81-0.80 (m, 38H, Ar—CH₂CH₂, NCCH₃ and phytyl chain);¹³C-NMR (CDCl₃, ppm): 11.77, 13.18, 14.28, 19.68, 19.73, 20.80, 20.96,22.62, 22.72, 23.00, 24.00, 24.41, 24.80, 25.37, 27.80, 32.77, 37.28,37.30, 37.74, 38.00, 39.00, 39.35, 54.69, 69.17 (aliphitic C), 125.11,126.55, 127.20, 130.63, 142.00, 149.60 (Ar—C), 173.62 (COOH); HRMS (CI,m/z): 502.426327 (M+H⁺, Calc. For C₃₂H₅₅NO₃ 502.426020).

6-(2,4-Dinitrophenylazo(2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-1,2,3,4-tetrahydroquinoline(43)

2,4-dinitrophenylhydrazine (1.95 g, 9.82 mmol) was dissolved in 34 mL ofhot absolute ethanol and 3.4 mL of concentrated sulfuric acid wascarefully added. The hot red solution was cooled to room temperature andthe iminoquinone (37, 1.00 g, 2.34 mmol) in 12 mL of absolute ethanolwas added with stirring. The mixture was allowed to stand at roomtemperature for 70 h, then diluted with 120 mL of water and extractedwith ether (3×100 mL). The combined ether extracts were washed neutralwith water (6×100 mL), dried (NaSO₄), and evaporated in vacuo to give2.9 g of a partly crystalline dark violet mass. This product waschromatographed over silica gel eluting with 5% ether in petroleum etherto yield 1.35 g (95%) of violet crystals (43), m.p. 72-73° C. (lit.¹73-75). HRMS (Cl, m/z): 608.417199 (M+H⁺, Calc. For C₃₅H₅₃N₅O₄608.417581).

EXAMPLE 3 Synthesis of 2,5,7,8-tetramethyl-2R-(4,8,12-trimethyl-3,7,11E:E tridecatrien)chroman-6-yloxy)acetic acid (44)

The starting material was 89% pure R,R,R,-alpha-tocotrienol obtained asa gift from Dr. Kai Baldenius, BASF Aktiengesellschaft, Ludwigshafen,Germany. The material was provided to Dr. Jeffrey Atkinson, BrockUniversity, Ontario, Canada who prepared 44, also named6-0-carboxymethyl alpha-tocotrienol or 6-0 CM-α-Toc, as a service. Thestarting material was subjected to column chromatography with a silicagel column (70-230 mesh, 60 A) using a step gradient ofhexane:dichloromethane (10:1, 5:1, 1:1). The recovered material was pureRRR-alpha-tocotrienol as determined by mass spectrometry, ¹H- and¹³C-NMR.

NaH (0.270 g of a 60% dispersion in mineral oil, 6.7 mmol) was suspendedin dry THF (75 mL) under an argon atmosphere at 0° C. for 10 min, atwhich time alpha-tocotrienol (2.00 g, 4.71 mmol) dissolved in 25 mL ofdry THF was added via cannula. This mixture was stirred at 0° C. for 15min under argon, then ethyl bromoacetate (0.93 g, 5.59 mmol) was addedvia syringe. The reaction was monitored by TLC (hexane:ethyl acetate,10:1, R_(f)=0.36) until it was judged complete after 3.5 hours. Thereaction solution was diluted with 50 mL of CH₂Cl₂, washed withsaturated NaCl solution (50 mL×3), dried over anhydrous Na₂SO₄ and thesolvent removed under reduced pressure. The crude product was purifiedby column chromatography on silica gel (70-200 mesh) using hexane:ethylacetate, 30:1) to yield 1.64 g (68%) of a clear oil.

The ester, ethyl 6-O-carboxymethyl-α-tocotrienol, (2.49 g, 4.87 mmol)was dissolved in 50 mL of dry THF, then 11.5 mL of 10% KOH was added andthe mixture stirred at room temperature for 6 hrs. The reaction progresswas monitored by TLC(CHCl₃:MeOH:CH₃CO₂H, 97:2.5:0.5, R_(f)=0.16). Theresulting solution was adjusted to pH 3 using 1N HCl and the productextracted with CH₂Cl₂ (50 mL×3), washed with saturated NaCl solution,dried over anhydrous Na₂SO₄, and the solvent removed under reducedpressure. The crude product was further purified by columnchromatography on silica gel (CH₂Cl₂:ethyl acetate, 9:1) to yield 1.85 g(79%) of a white waxy solid. ¹H-NMR (300 MHz) _(—)5.15 (m, 3H, HC═C),4.44 (s, 2H, COCH₂O), 2.61 (t, 2H, C-4 CH₂), J=7 Hz), 2.21 (s, 3H), 2.17(s, 3H), 2.14 (s, 3H), 2.16-1.90 (m, 10H), 1.82 (m, 2H, C-3 CH₂), 1.70(s, 3H, C-2 CH₃), 1.65 (overlapped multiplet, 2H), 1.62 (s, 911,C═CCH₃), 1.28 (s, 3H, CH₃). ¹³C-NMR (75 MHz, CDCl₃) 174.2, 148.9, 147.5,135.6, 135.4, 131.7, 127.8, 125.9, 124.8, 124.7, 124.6, 123.7, 118.2,77.6, 75.2, 69.6, 40.1, 31.6, 27.1, 27.0, 26.1, 24.2, 22.6, 21.0, 18.1,16.4, 16.3, 13.1, 12.3, 12.2. Only 30 of the potentially 31 distinctcarbon atoms are apparent in the spectrum. Two peaks, likely in thealiphatic region, are not resolved at this field strength. EI-MS m/z 482(M⁺, 100), 263 (15), 261 (11), 224 (15), 223 (97), 222 (10), 203 (6),165 (18), 137 (13), 95 (9), 81 (35), 69 (89)

EXAMPLE 4 Cell Culture Conditions

All test cell lines were cultured at 37° C. in 5% CO₂ in standard mediasupplemented with fetal calf serum, using established standardconditions. Plastic adherent cells were disassociated with trypsin,washed, counted, and used directly in experiments. All cells wereexamined routinely to verify no mycoplasma contamination.

EXAMPLE 5 Solubility and Dilution of Tocopherol and TocotrienolCompounds

All compounds were handled as if they were light sensitive(photodegradable). All compounds were initially dissolved in absoluteethanol and subsequently diluted to a final concentration of 0.5%ethanol with the appropriate media.

EXAMPLE 6

Determination of Effective Concentration (EC₅₀) of Naturally OccurringTocopherols and Tocotrienols

Table 1 provides tumor growth inhibitory properties of naturallyoccurring vitamin E compounds against the breast tumor cell lineMDA-MB-435. The naturally occurring α-, γ-, δ-tocotrienols exhibit tumorgrowth inhibitory properties with EC₅₀ of 40, 10, and 5 μM for DNAsynthesis arrest, and 50, 10, and 5 μM for apoptosis, respectively. Thenaturally occurring α, γ, and δ tocopherols were relatively ineffectiveas anticancer agents, with EC₅₀ for DNA synthesis arrest of >400, >400,and 150 μM, respectively, and EC50 for apoptosis of >400, 300, and 100μM, respectively (Table 1).

TABLE 1 EC₅₀ for DNA Synthesis Arrest and Apoptosis (μM) in MDA-MB-435DNA Synthesis Arrest Apoptosis Tocopherol α >400 >400 γ >400 300 δ 150100 Tocotrienol α 40 50 γ 10 10 δ 5 5

EXAMPLE 7 Determination of Effective Concentration (EC₅₀) of TocopherolDerivatives and Analogs to Induce Apoptosis

Whereas the parental non-structurally modified forms of tocopherols donot exhibit effective apoptotic properties against a battery of tumorcells, fifteen out of twenty-nine RRR-α-tocopherol compounds and two outof five 1-aza-α-tocopherol analogs, structurally modified via etherlinked moieties of different composition and size were extremelyeffective at inducing tumor cells to undergo apoptosis while having noapoptotic inducing properties on normal cells. Compounds 1, 2, 3, 7, 8,9, 12, 15, 17, 19, 20, 21, 22, 25, 26, 27, 39, and 42 exhibit effectivegrowth inhibitory (apoptotic inducing) properties specific for humancancer cells from a wide variety of cell lineages, including (i) breast(estrogen responsive Michigan Cancer Foundation human breast cancer cellline number 7, MCF-7 McGuire; non-estrogen responsive M.D. Andersonmetastatic breast human cancer cell line, MDA-MB-435; and, estrogennon-responsive M.D. Anderson metastatic human breast cancer cell line,MDA-MB-231); (ii) prostate (androgen responsive human prostate cancercell line, LnCaP and the androgen non-responsive human prostate cancercell line, PC-3 and the DU-145 cell line); (iii) promyelocytic leukemiacells (human Promyelocytic Leukemia Cell Line, HL-60), lymphoid celllines Jurkat and HL-60; (iv) cervical (human cervical cancer cell line,ME-180); (v) ovarian (human ovarian cancer cell line, C-170 cells); (vi)endometrial (human endometrial cancer cell line, RL-95-2 cells); (vii)colon cell lines DLD-1; and (viii) lung cell line A-549. Normal primarybreast cells (normal primary early passage human mammary epithelialcells, HMEC) and immortalized, non-tumorigenic mammary cells (MichiganCancer Foundation immortalized but non-tumorigenic human mammary number10A cells, MCF-10A) do not undergo apoptosis when cultured with theabove pharmacodynamically designed forms of tocopherol.

The apoptotic EC₅₀ for a battery of test cancer cells for thetwenty-nine novel RRR-α-tocopherol compounds and two of the five1-aza-α-tocopherol analogues of this invention are presented in Tables2-1/2-2 and 3-1/3-2.

TABLE 2-1 Cell Type VES 1 2 3 4 5 6 7 Breast Cancer HMEC N N N N N N N NMCF-10A N N N N N N N N MDA-MB-435  5-10  5-10 10-20  5-10 N N N  5-10MDA-MB-231  5-10  5-10 10-20  5-10 N N N  5-10 MCF-7 10-20  5-10 20-3020-30 N N N 10-20 T47D N N N N N N N N Cervical ME-180 10-20 1-5  5-1010-20 N N N  5-10 Ovarian C-170 N 10-20 10-20 10-20 N N N 10-20Endomerial RL-95-2 10-20 10-20  10-209 10-20 N N N  5-10 Prostate PREC NN NT NT NT NT NT NT LnCaP  5-10  5-10  5-10  5-10 N N N 2.5-5   PC-310-20  5-10  5-10  5-10 N N N  5-10 DU-145 10-20  5-10 NT NT NT NT NT NTColon HT-29  5-10 10-20 NT NT NT NT NT NT DLD-1 10-20 10-20 NT NT NT NTNT NT Lung A-549 20-30 10-20 NT NT NT NT NT NT Lymphoid Cells Myeloma10-20 NT NT NT NT NT NT NT Raji 10-20 NT NT NT NT NT NT NT Ramos 10-20NT NT NT NT NT NT NT Jurkat 10-20 10-20 NT NT NT NT NT NT HL-60 10-20 5-10 10-20 10-20 N N N  5-10

TABLE 2-2 Cell Type 8 9 10 11 12 13 14 15 Breast Cancer HMEC N N N N N NN NT MCF-10A N N N N N N N NT MDA-MB-435 5-10 5-10 N N  5-10 N N 20-30MDA-MB-231 5-10 5-10 N N  5-10 N N 20-30 MCF-7 10-20  10-20  N N  5-10 NN 20-30 T47D NT NT NT NT NT NT NT NT Cervical ME-180 5-10 5-10 N N  5-10N N N Ovarian C-170 10-20  N N N 10-20 N N N Endomerial RL-95-2 1-5 5-10 N N  5-10 N N N Prostate PREC NT NT NT NT NT NT NT NT LnCaP 5-105-10 N N >20-30  NT N NT PC-3 5-10 5-10 NT N 10-20 N N NT DU-145 NT NTNT NT NT NT NT NT Colon HT-29 NT NT NT NT NT NT NT NT DLD-1 NT NT NT NTNT NT NT NT Lung A-549 NT NT NT NT NT NT NT NT Lymphoid Cells Myeloma NTNT NT NT NT NT NT NT Raji NT NT NT NT NT NT NT NT Ramos NT NT NT NT NTNT NT NT Jurkat NT NT NT NT NT NT NT NT HL-60 10-20  10-20  N N 20-30 NN NT

TABLE 3-1 Cell Type 16 17 18 19 20 21 22 23 Breast Cancer HMEC NT NT NTNT NT NT NT NT MCF-10A NT N NT N N N NT N MDA-MB-435 N NT N 10-20 10-20N NT N MDA-MB-231 N NT NT NT NT NT NT NT MCF-7 N 10-20 N 10-20  5-10 N15-20 N T47D NT 10-20 NT N  5-10 NT NT NT Cervical ME-180 NT 20-30 N 1-51-5 1-5 NT NT Ovarian C-170 NT 20-30 N 1-5 * N NT NT Endomerial RL-95-2NT NT NT NT NT N NT NT Prostate PREC NT NT N NT NT NT NT NT LnCaP NT10-20 NT  5-10  5-10* N NT NT PC-3 NT NT NT N  5-10* N NT N DU-145 NT NTNT  5-10  5-10* N NT N Colon HT-29 NT N N NT NT NT NT N DLD-1 NT NT NTNT NT NT NT NT Lung A-549 NT N N 20-30 20-30 NT NT NT Lymphoid CellsMyeloma NT NT NT NT NT NT NT NT Raji NT NT NT NT NT NT NT NT Ramos NT NTNT NT NT NT NT NT Jurkat NT 10-20 N 10-20 10-20 NT NT NT HL-60 NT 10-20N 10-20 10 NT NT NT

TABLE 3-2 Cell Type 24 25 26 27 28 29 39 42 43 Breast Cancer HMEC NT NTNT NT NT NT NT NT NT MCF-10A NT N N NT NT NT NT NT NT MDA-MB- N N 20-40NT NT NT 10-20 10-20 PPT 435 MDA-MB- NT NT NT NT NT NT NT NT NT 231MCF-7 N N N 10-20 NT NT NT NT NT T47D NT NT N NT NT NT NT NT NT CervicalME-180 NT N * NT NT NT NT NT NT Ovarian C-170 NT N 20-30 NT NT NT NT NTNT NT Endomerial RL-95-2 NT N 20-30 NT NT NT NT NT NT Prostate PREC NTNT NT NT NT NT NT NT NT LnCaP N N 10-20 10-20 NT NT NT NT NT PC-3 N20-30 N NT NT NT NT NT NT DU-145 N 20-30 N NT NT NT NT NT NT Colon HT-29NT N N NT NT NT NT NT NT DLD-1 N N 20-40 NT NT NT NT NT NT Lung A-549 NTNT N NT NT NT NT NT NT Lymphoid Cells Myeloma NT NT NT NT NT NT NT NT NTRaji NT NT NT NT NT NT NT NT NT Ramos NT NT NT NT NT NT NT NT NT JurkatNT NT 20-30 NT NT NT NT NT NT HL-60 NT NT N NT NT NT NT NT NT

EXAMPLE 8 Determination of effective concentration (EC₅₀) of6-O-carboxymethyl alpha-tocotrienol

MDA-MB-435 and MCF-7 cells at 1.5×10⁵/well in 12 well plates forapoptosis and 1.5×10⁴/well in 96 well plates for DNA synthesis arrestwere incubated overnight in culture media and treated with α-tocopherol,α-tocotrinel, compound 1, 6-0-carboxymethyl alpha-tocotrienol(6-0-CM-α-T, 44), and vitamin E succinate (VES) at differentconcentrations in experimental media (2% serum for MDA-MB-435 cells and5% serum for MCF-7 cells). DNA synthesis arrest was determined by³H-uptake at one day treatment and apoptosis was detected by DAPIstaining at three day treatment. EC₅₀ was calculated as the dosage oftest compounds that gave 50% growth inhibition.

The compound, 6-0-carboxymethyl alpha-tocotrienol 44, has superioranti-tumor properties when compared to those of naturally occurringtocopherols and tocotrienols as well as vitamin E succinate or etherlinked succinated tocopherol-based compounds. Comparison of DNAsynthesis arrest properties and apoptotic inducing properties ofRRR-α-tocopherol, alpha-tocotrienol, compound 1, and 6-0-carboxymethylalpha-tocotrienol (6-0-CM-α-Toc, 44) are shown in Tables 4 and 5,respectively.

TABLE 4 EC₅₀ for DNA Synthesis Arrest (μM) Tumor Cell Lines CompoundsMDA-MB-435 MCF-7 α-tocopherol >100 >100 α-tocotrienol 29 40 Cmpd. 1 1520 6-0 CM-α-Toc (44) 2.5 5 VES 17 22

TABLE 5 EC₅₀ for Apoptosis (μM) Tumor Cell Lines Compounds MDA-MB-435MCF-7 α-tocopherol >100 >100 α-tocotrienol 66 40 Cmpd. 1 30 366-0-CM-α-Toc (44) 5.6 10 VES 36 40

The pharmacodynamically designed 6-0-carboxymethyl alpha-tocotrienol 44has an improved therapeutic index and is a potent inhibitor of cancercell growth; i.e., 6-0-carboxy methyl alpha-tocotrienol 44 exhibitsantitumor activity, as measured by DNA synthesis arrest, toward humanMDA-MB-435 and MCF-7 breast cancer cells 8-12 fold higher than theparent material, alpha-tocotrienol, 4-7 fold higher than vitamin Esuccinate, and 4-6 fold higher than compound 1. Also, 6-0-carboxymethylalpha-tocotrienol 44 is a potent apoptotic inducer when tested usinghuman MDA-MB-435 and MCF-7 breast cancer cells. 6-0-carboxymethylalpha-tocotrienol 44 induction of apoptosis for the two cell types was4-12 fold higher than alpha tocotrienol, 4-6 fold higher than vitamin Esuccinate and 4-5 fold higher than compound 1.

EXAMPLE 9 Bioassay for Apoptosis

Cells were cultured at 1.5×10⁵ cells/well in 12 well plates. Cells wereallowed to adhere overnight, then incubated with novel test compounds at0.01, 0.1, 1, 5, 10 & 20 μg/mL for 1, 2 and 3 days. After treatment,cells (floating+trypsin released adherent cells) were pelleted, washedand stained with 2 μg/ml DAPI (4′,6-diamidine-2′-phenylindoledihydrochloride) in 100% methanol for 15 minutes at 37° C. and/or 3′-OHends resulting from DNA fragmentation were detected with the TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP-X 3′ nickend-labeling) technique, and then viewed using a Zeiss ICM 405microscope. Cells whose nucleus contained clearly condensed orfragmented chromatin or stained for fragmented DNA in the TUNEL assaywere scored as apoptotic. Data are presented as percent cells undergoingapoptosis.

EXAMPLE 10 Bioassay for DNA Synthesis Arrest

To assay DNA synthesis, all cells were used at 2.5×10⁵/ml. Cells weretreated with each of the compounds at concentrations of 0.01, 0.1, 1, 5,10 and 20 μg/mL and 200 μl of each treatment group were plated inquadruplicate in a 96 well culture plate (Corning, Corning N.Y.).Experiments were done in duplicate, one plate used for viability testingand the other plate for examination of ³H-TdR uptake to monitor DNAsynthesis. Plates were cultured for 48 hours at 37° C., 5% CO₂. Eighthours prior to the end of incubation, ³H-TdR was added to one of theduplicate plates and incubation continued for 8 hours.

The cells were then harvested (trypsinization was required to harvestadherent cells), and isotope uptake was determined as counts per minute(cpm). For viability studies, at the end of the incubation, the cellswere removed from the wells and viability checked by the Trypan BlueExclusion method. Percent viability and percent DNA synthesis incomparison to untreated or vehicle treated cells of each treatment groupwere calculated.

EXAMPLE 11 Bioassay for Cell Cycle Arrest

The cells were cultured with novel test agents for 2-3 days, fixed in95% ethanol and stained with propidium iodide overnight. DNA content wasdetermined using a Coulter Epics Elite Flow Cytometer with an argonlaser setting of 488 nm. Cell size was measured simultaneously, and datawere analyzed as to percent cells in each cell cycle phase using theCoulter Multicycle Program.

EXAMPLE 12 Bioassay for Cellular Differentiation

To determine if the novel compounds were inducing cellular morphologicalfeatures of differentiation, the cells were cultured on cover slips,then unstained cells were examined for cell shape and nuclear versuscytoplasmic volumes. Cells were also fixed with 4% paraformaldehyde andstained with Oil Red O for detection of milk lipids. Additionally, cellswere examined by Western immunoblot analyses for presence of variousbiomarkers of differentiation including: Her-2/neu, p21 Waf2/Cip1, andcytokeratin 18, and examined by RT-PCR for presence of β-casein mRNA.

EXAMPLE 13 Mechanisms of Induction of Apoptosis

Without being limited by theory, the mechanism of induction of apoptosisby these compounds appears to involve three distinct apoptotic signalingpathways; namely, activation of latent transforming growth factor-beta(TGF-β), activation of the Fas/Fas ligand signaling pathways, andsignaling by the stress kinase (c-Jun N-terminal Kinase) pathway.

TGF-βs are potent growth inhibitory molecules that are known to inhibitcell growth by inhibition of DNA synthesis arrest and by induction ofapoptosis. TGF-βs are involved only in induction of the apoptoticpathway, i.e., there is no evidence currently that the TGF-βs effect DNAsynthesis arrest; however, this possibility has not been completelyruled out. TGF-βs are made and secreted by cells in a latent non-activeform. To be effective as tumor growth inhibitors, the latent TGF-βs mustbe activated by induction of cell surface proteins that provide a properstructure for processing and activating proteases that cut the latentprotein and release the active TGF-β.

The compounds of the present invention are shown to activate proteasessuch as cathepsin D family proteases, and upregulate themannose-6-phosphate receptor which binds inactive TGF-β and permitsactivation via proteases. Active TGF-β signals via cell membrane TGF-βreceptors I and II to activate down stream kinases referred to as stresskinases or c-Jun N-terminal Kinases (JNK) which phosphorylate andactivate transcription factors c-Jun, ATF-2 and Elk-1. Prolongedactivation of transcription factor c-Jun causes tumor cells to undergoapoptosis. These transcription factors, acting as homodimers orheterodimers with a multitude of transcription factor partners activateproapoptotic genes and/or down-regulate antiapoptotic genes leading toDNA fragmentation. The compounds of the present invention do notgenerate an anti-proliferative outcome to TGF-β signaling in normalnon-tumor cells.

A second apoptotic inducing mechanism called the Fas/Fas ligandapoptotic signaling pathway is activated by the novel compounds of thepresent invention. Activated Fas/Fas ligand signaling may lead to rapidcell death by apoptosis. Thus, for tumor cells to escape death byFas/Fas ligand, they must inactivate this most important apoptoticpathway. The mechanism for inactivation of the Fas/Fas ligand signalingpathway by tumor cells varies; however, many tumor cells down regulatethe expression of Fas receptor and Fas ligand on their membranes.

Most important,R,R,R-2-(2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yloxy)aceticacid (1) has been shown to induce Fas/Fas ligand resistant tumor cellsto become Fas/Fas ligand sensitive. Compound 1 also has the ability toenhance the expression of Fas ligand on the membrane of LNCaP prostatecells. Studies show that Fas signaling resistant human breast cancercells retain the Fas receptor in their cytoplasm, but when cultured withcompound 1, the Fas receptor is transported from the cytoplasm to themembrane; thereby rendering the cells Fas signaling sensitive.Furthermore, this compound is synergistic in anti-Fas triggeredapoptosis in that greater amounts of cell killing is obtained with bothhuman breast and prostate cancer cells when co-treated versus whentreated separately. The ability of compound 1 to convert Fas signalingresistant tumor cells to Fas signaling sensitive tumor cells and toexhibit synergistic killing effects provides an extremely importantmechanism for destruction of tumor cells both by the host immunesurveillance system as well as by pharmaceutical intervention. Thecompounds of the present invention do not activate the Fas signalingpathway of normal non-tumor cells.

These compounds activate the JNK kinase signaling pathway, perhaps byTGF-β and Fas/Fas ligand signaling. Prolonged activation of JNK resultsin prolonged activation of c-Jun and ATF-2 transcription factors, whichare postulated to play a role in expression or repression ofproapoptotic and antiapoptotic genes, respectively.

EXAMPLE 14 Mechanism of Induction of DNA Synthesis Arrest, Cell CycleArrest and Cellular Differentiation

The mechanisms of growth inhibition by DNA synthesis arrest, cell cyclearrest and by induction of cellular differentiation have not beencharacterized as fully as the mechanism of growth inhibition byapoptosis. Studies show that the compounds of the present invention haveprofound effects on the cell cycle, inducing DNA synthesis arrest ofapproximately 95% of the tumor cells within 24 hours of treatment. Tumorcells cultured with the compounds disclosed herein are growth inhibitedin the G1 cell cycle phase, undergo morphological changes and expressmilk lipids, an indication that the cell cycle blocked cells haveundergone differentiation. P21, a gene known to be an inhibitor ofentrance of cells from the G1 cell cycle phase to the S phase of thecell cycle, and the mRNA, as well as the protein of P21 gene, isup-regulated by treatment of MDA-MB-435 human breast cancer cells withcompound 1.

EXAMPLE 15 In Vivo Potential for Human Cancer Cells

The present invention has potential for use as therapeutic agents. Invivo studies of tumor growth and metastasis of human tumor cells eitherectopically or orthotopically transplanted into immune compromisedanimals, such as nude mice, or in vivo studies employing well recognizedanimal models are conducted. Inhibition of growth of human tumor cellstransplanted into immune compromised mice, provide pre-clinical data forclinical trials. In vivo studies include two human tumor cell models,the metastatic non-estrogen responsive MDA-MB-435 breast cancer model,the androgen non-responsive PC-3 prostate cancer model and a murinesyngeneic 66c1.4-GFP mammary cancer model.

MDA-MR-435 Breast Cancer Model:

Pathogen free MDA-MB-435 human breast cancer cells stably transfectedwith a marker protein (fluorescent green protein) are grown as a solidtumor in immune compromised nude or SCID mice. 1×10⁶ tumor cells areorthotopically injected into the mammary fat pad or ectopically injectednear the 4th and 5th nipples of female nude mice. Tumor growth,metastasis, and death of the animals are determined. Tumor growth ismeasured by caliper evaluations of tumor size. At the time of sacrifice,tumors are removed, measured for size, and used for histochemicalexamination. Organs such as spleen, lymph nodes, lungs, and bone marrow,are examined for metastatic MDA-MB-435 cells by histochemical stainingof tissue sections for expression of the marker fluorescent greenprotein.

PC-3 Prostate Cancer Model

Pathogen free PC-3 human prostate cancer cells stably transfected with amarker protein (fluorescent green protein) are grown as a solid tumor innude mice. The tumors are removed, and 1 mm sections of equal size areectopically transplanted into the hind flank of male nude mice. Tumorgrowth, metastasis, and death of the animals are determined. At the timeof sacrifice, tumors are removed, measured for size, and used forhistochemical examination. Organs such as spleen, lymph nodes, lungs,bone marrow, are examined for metastatic PC-3 cells by histochemicalstaining of tissues for expression of the marker fluorescent greenprotein.

Murine Syngeneic 66c1.4-GFP Mammary Cancer Model

Pathogen free 66c1.4-GFP mammary cancer cells of Balb/c origin (100,000to 200,000) are injected near the 4th and 5th nipples of female Balb/cmice. Tumor metastases to lungs occur in 100% of the mice. Tumor growth,metastasis, and death of the animals are determined. Tumor growth ismeasured by caliper evaluations of tumor size. At the time of sacrifice,tumors are removed, measured for volume, and used for histochemicalexamination. Organs such a s spleen, lymph nodes, lungs, and bonemarrow, are examined for metastatic cells by histochemical staining oftissue sections for expression of the marker green fluorescence protein.

Skin Cancer Animal Model

Skin cancer is induced in SENCAR and SKH-1 hairless mice by ultravioletirradiation and chemical (DMBA) treatments. In addition, micespecifically expressing the oncogene Her-2/neu in skin basal cells thatspontaneously develop skin cancer are used. The compounds disclosedherein are topically applied to the skin daily, before and after skincancer initiation, and development of skin papilloma formation isassessed. Control mice are treated identically except that they receivevehicle treatments topically applied to their skin. The efficacy ofthese compounds in treating papilloma's as well as their ability toaffect malignant conversion when supplied prior to premalignantprogression is monitored.

EXAMPLE 16 Supplementation with Novel Compounds

Prior to initiation of the in vivo experiments, the compounds of thisinvention that exhibit the greatest amount of tumor cell killing areadminstered to nude, SCID, transgene, and other mice at varying levelsto establish the highest level of compound that can be administeredsafely without adverse effects. The compounds are administered in amodel-appropriate manner; e.g., liposomal/aerosol/nasal/lungs, orally,injections, including injections directly into the target organ, ortopically. After establishing the highest level of the compounds thatcan be tolerated and effective administration routes, the novelcompounds are administered to the mice on a daily basis, and tumorgrowth and progression is determined as described above.

EXAMPLE 17 Establishing Maximum Tolerated Dose (MTD) of2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)aceticacid (1)

Prior to conducting the preclinical chemotherapeutic studies, compound 1is assayed in non-tumor Balb/c female mice in an effort to establish themaximum tolerated dose. Compound 1 is dissolved in 100% ethanol toestablish a stock solution (2 grams of compound 1 in 5 mls of 100%ethanol) and diluted in peanut oil (non vitamin E supplemented) to yield20, 10, and 5 mg/0.1 ml. RRR-α-tocopherol succinate (VES; the succinatemoiety is attached to RRR-α-tocopherol via an ester linkage) wassolubilized and diluted as described above to yield 20 mg/0.1 ml/gavage,serving as an additional control. A total of 30 Balb/c female mice wereplaced into 6 groups (5 mice/group) and supplemented by gavage withcompound 1 daily for a total of 23 days. Group 1 mice were non treatedand served a s control; group 2 mice were the vehicle control group andreceived 0.1 ml of vehicle (ethanol+peanut oil equivalent to treatmentgroups)/mouse/day for a total of 23 days, group 3 mice received 20 mg/mlof VES daily for a total of 23 days. Treatment groups 3, 4 and 5 weregavaged daily for 23 days with 5, 10, and 20 mgs of compound 1. Compound1 at 5, 10, and 20 mg/day, administered daily by gavage to mice ingroups 3, 4, and 5, respectively, for 23 days is tolerated well by themice. Weights are determined weekly and there is no significantdifference in the weights of the mice receiving compound 1 at 5, 10, and20 mg/day when compared with the weights of the VES treated, untreatedand vehicle treated control mice (FIG. 7, data is for days 11 and 23days treatment). Mice remained active and showed no signs of toxicity.At the completion of the MTD studies, mice were sacrificed and noevidence of toxicity was observed upon autopsy.

EXAMPLE 18 Chemotherapeutic effectiveness of2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)aceticacid (1)

The maximum tolerated dose studies show that compound 1 administered atthe highest level (20 mg/day/23 days) is not toxic; however, due to thenon-toxic levels used, an maximum tolerated dose was not established.For the preclinical chemotherapeutic studies, in the absence of anmaximum tolerated dose, the mice are supplemented with 30 and 20 mgs/0.1ml by gavage daily for 21 days. Compound 1 is dissolved in 100% ethanolat 2 grams of compound 1/5 mls of ethanol, and diluted in peanut oil(nonsupplemented vitamin E) to yield 30 and 20 mgs of compound 1 per 0.1ml. Vehicle control consisted of a mixture of ethanol and peanut oilequivalents. The preclinical chemotherapeutic studies are conducted withcompound 1, utilizing human MDA-MB-435 human breast cancer cells, humanDU-145 prostate cancer cells, and human HT-20 colon tumor cellstransplanted into Balb/c immune compromised nude mice.

A total of 40 immune compromised nude mice are utilized for testing eachtest compound (two treatment groups at 20 and 30 mg/day) in each tumorcell type. Four experimental treatment groups of 10 mice each(vehicle/0.1 ml/gavage daily for 21 days, compound 1 at 30 mgs ofcompound #1/0.1 ml gavage/daily for 21 days, and compound 1 at 20 mgs/ofcompound 1/0.1 ml gavage daily for 21 days, for each tumor type areused. An established dosage of effective chemotherapeutic drugs is usedin the xenograft studies. Taxol at 20/mg/kg, administeredintraperitioneally daily for 5 days, is used for a positive control forhuman MDA-MB-435 breast cancer xenografts; Mitoxantrone at 1 mg/kg,administered daily intravenously for 5 days, is used as the positivecontrol for human DU-145 prostate cancer cells; and 5 Fluorouracil (5FU) at 30 mg/kg, administered daily for 5 days, is used for a positivecontrol for human HT-29 colon cancer cells. Tumor cells are transplantedsubcutaneously into the left flank and permitted to grow toapproximately 70 mg in size, test compound treatments by daily gavageare initiated, and continue for a total of 21 days. Mice are monitoreddaily for tumor size by caliper measurements. Mice are treated for 21days; however, the mice are sacrificed when the tumors of the vehiclecontrol reached 500 mg in weight. At the completion of the protocol,tumors are excised and tumor size established by weight. At the time ofsacrifice, serum, tumor, and heart muscle are taken from animals in thetwo compound 1 test groups in order to establish levels of compound 1via HPLC analyses (data not included). The chemotherapeuticeffectiveness of compound 1 administered daily at 30 and 20 mgs/day isdetermined by comparing the tumor size of the two treatment groups withthe vehicle control group for each of the three tumor types, and bycomparing the size of the tumor in the two treatment groups with thetumor group from the positive control. Treatment is for 21 days,experiments are continued until mean weight of tumor for vehicle controlreaches 500 mgs.

The in vivo preclinical chemotherapeutic data are depicted in Tables 3and 4 and FIG. 8A-8C and are presented for 21-22 days of treatment.Treatment of nude mice transplanted with human MDA-MB-435 breast cancercells with compound 1 for 21 days at 20 and 30 mg/day by gavage reducedthe mean tumor weight (mg) of the transplanted tumor cells by 20.7 and44.6%, respectively when compared to the mean tumor weight of thevehicle control group at day 21 after treatment. The positive control,Taxol, reduces the mean tumor growth, when compared to mean tumor growthof vehicle control group, by 90.3% (Table 6, 7 & FIG. 8A). Data areconverted to percent mean tumor growth in relation to weight gain at thebeginning of treatment (day 1) and are depicted in Table 7. Treatment ofnude mice transplanted with human DU-145 prostate cancer cells withcompound 1 for 21 days at 20 and 30 mg/day by gavage reduces the meantumor weight (mg) of the transplanted tumor cells by 31.5 and 24.8%,respectively, when compared to the mean tumor weight of the vehiclecontrol group at day 22 (Table 6, 7 & FIG. 8B). The positive control,mitoxantrone reduces the mean tumor growth, when compared to mean tumorgrowth of vehicle control group, by 27.9%. Treatment of immunecompromised nude mice transplanted with human HT-29 human colon cancercells with compound 1 for 21 days at 20 and 30 mg/day by gavage reducesthe mean tumor weight (mg) of the transplanted tumor cells by 33.3 and34.5%, respectively when compared to the mean tumor weight of thevehicle control group at day 22 (Table 6, 7 & FIG. 8C). The positivecontrol, 5 Fluorouracil, reduces the mean tumor growth, when compared tomean tumor growth of vehicle control group, by 31.4%.

TABLE 6 MDA-MB-435 Human Breast Cancer Cells Transplanted into Nude MiceMean tumor Weights (mg) Following Treatments Treatments 1 4 7 10 14 1721 Vehicle 70.1 98.2 119.5 153.2 172.8 203.7 226.1 #1 (20 mg) 70.1 85.8123.9 127.9 147.2 143.8 179.4 #1 (30 mg) 68.1 85.7 97.3 98.4 126.6 118.7125.2 Taxol 70.7 71.5 38.2 11.7 5.7 3.6 6.8 DU-145 Human Prostate CancerCells Transplanted into Nude Mice Mean tumor Weights (mg) FollowingTreatments Treatments 1 5 8 12 15 19 22 Vehicle 66.5 111.4 167.1 258.5351.1 458.3 540.9 #1 (20 mg) 66.5 103.5 137.9 208 254.8 362.2 370.5 #1(30 mg) 67.8 102.5 131.3 241.2 256.8 379.9 407 Mitoxantrone 66.8 107.2141.1 185.4 205.2 325.3 389.4 HT-29 Human Colon Cancer CellsTransplanted into Nude Mice Mean tumor Weights (mg) Following TreatmentsTreatments 1 5 8 12 15 19 22 Vehicle 80.3 91.7 133.2 185.6 204.8 260.1372.2 #1 (20 mg) 59.5 84.0 97.0 124.6 147.3 198.0 248.2 #1 (30 mg) 40.083.0 96.7 129.3 144.1 176.1 243.8 5-FU 59.3 87.5 105.8 145.8 141.6 212.3255.5

TABLE 7 Percent mean tumor weight gain (mg) following treatments¹MDA-MB-435 Human Breast Cancer Cells Treatments 1 4 7 10 14 17 21Vehicle 100 140.1 170.5 218.5 246.5 290.6 322.5 #1 (20 mg) 100 122.4176.7 182.5 210.0 205.1 255.9 #1 (30 mg) 100 125.8 142.9 144.5 185.9174.3 178.6 Taxol 100 101.1 −46.0 −83.7 −91.8 −99.5 −90.3 DU-145 HumanProstate Cancer Cells Treatments 1 5 8 12 15 19 22 Vehicle 100 167.5251.3 388.7 528.0 458.3 689.2 #1 (20 mg) 100 155.6 207.4 312.8 383.2362.2 544.7 #1 (30 mg) 100 151.2 193.7 355.8 378.8 379.9 560.3Mitoxantrone 100 160.5 211.2 277.5 307.2 487.0 582.9 HT-29 Human ColonCancer Cells Treatments 1 5 8 12 15 19 22 Vehicle 100 152.1 220.9 307.8339.6 431.3 617.2 #1 (20 mg) 100 141.2 163.0 209.4 247.6 332.8 417.1 #1(30 mg) 100 139.5 162.5 217.3 242.2 296.0 409.7 5-FU 100 147.6 178.4245.9 247.2 358.0 430.0 ¹Percent Mean Tumor Weight Gain (mg) followingtreatment was determined by dividing the mean tumor weight at varioustime periods following treatment by the mean tumor weight at day 1within the same treatment group and multiplying by 100.

EXAMPLE 19 Preparation of stock solution, vehicle and2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)aceticacid (1) dilutions

Compound 1 was prepared weekly, and stored at 4° C. The preparationprocedures are as follows:

Stock Solution of Compound 1:

Dissolve 2 grams of compound 1 in 5 mls of 100% ethanol (ETOH) andvortex at 37° C. This is the maximum amount of compound 1 that will gointo solution.

For the following dilutions, dry compound 1 is added to yield theappropriate levels of compound 1 while keeping the ethanol levels equalin the two experimental groups and the vehicle control group.

Compound 1 at 30 mg/0.1 ml Gavage/Mouse:

Combine 1 ml of compound 1 stock solution, 3 mls of vitamin E depletedpeanut oil and 800 mg of compound #1 (dry) and vortex at 37° C.

Compound 1 at 20 mg/0.1 ml Gavage/Mouse:

Combine 1 ml of compound 1 stock solution, 3 mls of vitamin E depletedpeanut oil and 400 mg of compound #1 (dry) and vortex at 37° C.approximate 2 h until in solution.

Compound 1 at 10 mg/0.1 ml gavage/mouse:

Combine 1 ml of compound 1 stock solution and 3 mls of vitamin Edepleted peanut oil.

Vehicle:

Combine 1 ml ETOH and 3 mls of vitamin E depleted peanut oil.

EXAMPLE 20 Chemopreventive properties of2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy)aceticacid (1) in an ACT rat cancer model

Compound 1 is used in vivo to treat transplanted human breast, prostate,and colon tumors transplanted in immune compromised nude mice. Thechemopreventive effectiveness of compound 1 in vivo against human breastcancer is shown in an estrogen cancer initiated ACI rat breast cancermodel. Approximately 90% of rats implanted with estrogen pellets developbreast cancer within 6 months after estrogen implantation.

Compound 1 is dissolved in 100% ethanol and is diluted to theappropriate dosage using vitamin E depleted peanut oil. The maximumtolerated dose (MTD, maximum dose of compound that can be administeredwithout adverse affects) is determined as described in Examples 14 and15. Compound 1 is administered at the maximum tolerated dose and 50% ofthe maximum tolerated dose. ACI rats at 4 weeks of age aresubpannicularly implanted with estrogen pellets in the shoulder region.Compound 1 at maximum tolerated dose and 50% of the maximum tolerateddose is administered by gavage. Breast tumors are detected in thecontrol group at approximately 100 days following estrogen implantation.Ninety percent of the control rats develop breast cancer within 6 monthsafter estrogen implantation. Tumor bearing animals from control andtreatment groups are sacrificed at various time intervals aftertreatment initiation, and mammary tissue is examined for obvious tumors,and further examined by histological analyses.

The following references are cited herein.

1. Colowisk, Sidney, P. and Kaplan, Nathan, O. Methods in Enzymology,Vol. XVIII, Vitamins and Coenzymes, Part C. Edited by Donald B.McCormick and Lemuel D. Wright. Section XII, PP. 335.

2. Dhar, A., Liu, S., Klucik, J., Berlin, K. D., Madler, M. M., Lu, S.,Ivey, R. T., Zacheis, D., Brwon, C. W., Nelson, E. C., P. J., Benbrook,D. M. Synthesis, Structure-Activity Relationships, and RARγ-LigandInteractions of Nitrogen Heteroarotinoids. J. Med. Chem. 42, pp.3602-3614 (1999).

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually incorporated byreference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

What is claimed is:
 1. A pharmaceutical composition comprising acompound of the formula:

wherein: R₁ is hydroxy or methoxy; R₂ and R₃ are independently hydrogenor methyl, and a pharmaceutically acceptable carrier.
 2. Thepharmaceutical composition of claim 1, wherein R₁ is hydroxy.
 3. Thepharmaceutical composition of claim 1, wherein R₁ is methoxy.
 4. Thepharmaceutical composition of claim 1, wherein R₂ is hydrogen.
 5. Thepharmaceutical composition of claim 1, wherein R₂ is methyl.
 6. Thepharmaceutical composition of claim 1, wherein R₃ is hydrogen.
 7. Thepharmaceutical composition of claim 1, wherein R₃ is methyl.
 8. Thepharmaceutical composition of claim 1, wherein the compound is


9. The pharmaceutical composition of claim 1, wherein the compound is